Antisense modulation of phospholipase a2, group vi (ca2+independent) expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Phospholipase A2, group VI (Ca2+-independent). The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Phospholipase A2, group IV (Ca2+-independent). Methods of using these compounds for modulation of Phospholipase A2, group VI (Ca2+-independent) expression and for treatment of diseases associated with expression of Phospholipase A2, group VI (Ca2+-independent) are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of Phospholipase A2, group VI (Ca2+-independent). In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Phospholipase A2, group VI (Ca²⁺-independent). Such compounds have been shown to modulate the expression of Phospholipase A2, group VI (Ca2+-independent).

BACKGROUND OF THE INVENTION

[0002] The enzymes of the phospholipase A2 (PLA2) family catalyze hydrolysis of the sn-2 fatty acid bond of phospholipids to liberate free fatty acids and lysophospholipids. These metabolites are involved in diverse cellular processes including signal transduction, host defense (including antibacterial effects), formation of platelet activating cofactor, membrane remodeling and general lipid metabolism (Dennis, J. Biol. Chem., 1994, 269, 13057-13060; Dennis, Trends Biochem. Sci, 1997, 22, 1-2). While the human PLA2 enzymes are of greatest interest, most of the current understanding of PLA2s has been obtained by studying enzymes from non-human sources. Since most of the human PLA2s have essentially identical non-human counterparts, the knowledge obtained from the non-human enzymes is equally applicable to the human enzymes (Dennis, J. Biol. Chem., 1994, 269, 13057-13060).

[0003] PLA2s are a diverse class of enzymes with regard to function, localization, regulation, mechanism, structure and dependence on divalent metal ions for activity (Dennis, J. Biol. Chem., 1994, 269, 13057-13060; Dennis, Trends Biochem. Sci, 1997, 22, 1-2). The PLA2s have been divided into ten groups (denoted using Roman numerals I through X) (Cupillard et al., J. Biol. Chem., 1997, 272, 15745-15752; Dennis, Trends Biochem. Sci, 1997, 22, 1-2). The PLA2s of groups IV and VI are intracellular, high molecular weight enzymes which have not been as extensively studied as the secreted PLA2s (groups I-III, V and X) (Balsinde and Dennis, J. Biol. Chem., 1997, 272, 16069-16072.). Group IV requires calcium for activity whereas the activity of group VI is calcium-independent.

[0004] First cloned from Chinese hamster ovary cells in 1997 (Tang et al., J. Biol. Chem., 1997, 272, 8567-8575.), the calcium-independent phospholipase A2 (PLA2 group VI, also known as iPLA2 and PLA2G6) plays a housekeeping role in phospholipid remodeling (Balsinde and Dennis, J. Biol. Chem., 1997, 272, 16069-16072.). Signaling roles in generation of substrate for leukotriene biosynthesis (Larsson Forsell et al., FEBS Lett., 1998, 434, 295-299.), generation of lipid messengers involved in regulation of ion channel activity (Ma et al., J. Biol. Chem., 1997, 272, 11118-11127.) and apoptosis (Atsumi et al., J. Biol. Chem., 1998, 273, 13870-13877.) have also been proposed for this enzyme.

[0005] Human calcium-independent phospholipase A2 (PLA2 group VI) expressed in B-cells was found to undergo extensive alternative splicing, generating multiple isoforms that contribute to a novel catalytic control mechanism (Larsson et al., J. Biol. Chem., 1998, 273, 207-214.). In addition, Ma et al. have mapped human calcium-independent phospholipase A2 (PLA2, group VI) to chromosome 22q13.1 and identified mRNA encoding two catalytically active isoforms in pancreatic islet cells. The long 88 KDa isoform is known as LH-iPLA2 while the shorter, 85 KDa protein (SH-iPLA2) is a splice variant produced by an exon-skipping mechanism (Ma et al., J. Biol. Chem., 1999, 274, 9607-9616.).

[0006] Functional impairment of pancreatic islet beta cells is implicated in both type I and type II diabetes mellitus (Ma et al., J. Biol. Chem., 1999, 274, 9607-9616.). In rodent islets, calcium-independent phospholipase A2 (PLA2 group VI) has been proposed to play a signaling role in glucose-induced insulin secretion (Tang et al., J. Biol. Chem., 1997, 272, 8567-8575.) and in experimentally induced beta cell apoptosis (Zhou et al., J. Clin. Invest., 1998, 101, 1623-1632.).

[0007] A number of small molecules have been employed as inhibitors of calcium-independent phospholipase A2 (PLA2 group VI), including: arachidonyl trifluoromethyl ketone, arachidonyl tricarbonyl and methyl arachidonyl fluorophosphonate which function as transition state analogues and bromoenol lactone which acts an irreversible mechanism-based inhibitor (Balsinde and Dennis, J. Biol. Chem., 1997, 272, 16069-16072.).

[0008] An antisense phosphorothioate oligonucleotide targeting nucleotides 59-78 of murine calcium-independent phospholipase A2 (PLA2 group VI) mRNA was used to inhibit the process of phospholipid fatty acid remodeling in murine P388Dl macrophages (Balsinde et al., J. Biol. Chem., 1997, 272, 29317-29321.).

[0009] Pharmacological modulation of calcium-independent phospholipase A2 (PLA2 group VI) activity and/or expression may be an appropriate point for therapeutic intervention in pathologic conditions such as diabetes mellitus types I and II and conditions related to abnormal apoptosis. To date, investigative strategies aimed at inhibiting the action of calcium-independent phospholipase A2 (PLA2 group VI) have been limited to the previously cited studies involving small molecule inhibitors and the single antisense oligonucleotide. Consequently, there remains a need for additional agents which modulate the function of this enzyme.

[0010] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of calcium-independent phospholipase A2 (PLA2 group VI) expression.

[0011] The present invention provides compositions and methods for modulating calcium-independent phospholipase A2 (PLA2 group VI) expression, including modulation of the splice variant forms of calcium-independent phospholipase A2 (PLA2 group VI).

SUMMARY OF THE INVENTION

[0012] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Phospholipase A2, group VI (Ca2+-independent), and which modulate the expression of Phospholipase A2, group VI (Ca2+-independent). Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Phospholipase A2, group VI (Ca2+-independent) in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Phospholipase A2, group VI (Ca2+-independent) by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Phospholipase A2, group VI (Ca2+-independent), ultimately modulating the amount of Phospholipase A2, group VI (Ca2+-independent) produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Phospholipase A2, group VI (Ca2+-independent). As used herein, the terms “target nucleic acid” and “nucleic acid encoding Phospholipase A2, group VI (Ca2+-independent)” encompass DNA encoding Phospholipase A2, group VI (Ca2+-independent), RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Phospholipase A2, group VI (Ca2+-independent). In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0014] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Phospholipase A2, group VI (Ca2+-independent). The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or-codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Phospholipase A2, group VI (Ca2+-independent), regardless of the sequence(s) of such codons.

[0015] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0016] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation-codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0017] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0018] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0019] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0020] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.

[0021] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0022] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0023] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0024] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16;

[0025] Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0026] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0027] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0028] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0029] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0030] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0031] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0032] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0033] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0034] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0035] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. No.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0036] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0037] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0038] A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0039] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0040] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′, 2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0041] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0042] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine; folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO. J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in United States Patent Application 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0043] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0044] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0045] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0046] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0047] The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.

[0048] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0049] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0050] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0051] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0052] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0053] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0054] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Phospholipase A2, group VI (Ca2+-independent) is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0055] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Phospholipase A2, group VI (Ca2+-independent), enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Phospholipase A2, group VI (Ca2+-independent) can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Phospholipase A2, group VI (Ca2+-independent) in a sample may also be prepared.

[0056] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0057] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0058] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.

[0059] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0060] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0061] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0062] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0063] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0064] Emulsions

[0065] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

[0066] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0067] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0068] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0069] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0070] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0071] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0072] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0073] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in ah aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0074] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0075] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0076] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0077] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0078] Liposomes

[0079] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0080] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0081] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0082] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0083] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0084] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0085] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0086] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0087] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0088] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0089] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0090] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0091] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0092] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses iiposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0093] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0094] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0095] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0096] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0097] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0098] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0099] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0100] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0101] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0102] Penetration Enhancers

[0103] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0104] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile-salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0105] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252)

[0106] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0107] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharnacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0108] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0109] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0110] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0111] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0112] Carriers

[0113] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0114] Excipients

[0115] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0116] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0117] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0118] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0119] Other Components

[0120] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0121] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0122] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th-Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0123] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0124] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in-maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0125] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0126] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites

[0127] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

[0128] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

[0129] 2′-Fluoro amidites

[0130] 2′-Fluorodeoxyadenosine amidites

[0131] 2′-fluoro oligonucle6tides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0132] 2′-Fluorodeoxyguanosine

[0133] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0134] 2′-Fluorouridine

[0135] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0136] 2′-Fluorodeoxycytidine

[0137] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0138] 2′-O-(2-Methoxyethyl) modified amidites

[0139] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

[0140] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0141] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

[0142] 2′-O-Methoxyethyl-5-methyluridine

[0143] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

[0144] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0145] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

[0146] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0147] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

[0148] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0149] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

[0150] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0151] A solution of 3′-O-ace tyl-2′-O-methoxyethyl-5′-O-dimethoxy-trityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH3 gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

[0152] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine

[0153] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

[0154] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dinethoxytrityl-5-methyl-cytidine-3′-amidite

[0155] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)-phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound. 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0156] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0157] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0158] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0159] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, l19.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

[0160] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0161] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160 ° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

[0162] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0163] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

[0164] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0165] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂Cl₂ and the combined organic phase was washed with water, brine and dried over anhydrous Na₂SO₄. The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0166] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0167] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated to dryness . The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂Cl₂ to get 5′-O-tert-butyldiphenylsilyl-2′O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

[0168] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0169] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

[0170] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0171] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (111 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0172] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0173] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P₂O₅ under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate. (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

[0174] 2′-(Aminooxyethoxy) nucleoside amidites

[0175] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0176] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0177] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0178] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0179] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O-CH₂-O-CH₂-N(CH₂)2, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0180] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0181] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.

[0182] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl uridine

[0183] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃ solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the title compound. 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0184] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.

Example 2

[0185] Oligonucleotide Synthesis

[0186] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

[0187] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0188] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0189] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0190] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0191] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0192] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0193] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0194] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0195] Oligonucleoside Synthesis

[0196] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0197] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0198] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0199] PNA Synthesis

[0200] Peptide nucleic acids (PNAS) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0201] Synthesis of Chimeric Oligonucleotides

[0202] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0203] [2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

[0204] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0205] [2′-O-(2-Methoxyethyl)]-[2′-deoxyl]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0206] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0207] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0208] [21-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0209] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0210] Oligonucleotide Isolation

[0211] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al.; J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0212] Oligonucleotide Synthesis—96 Well Plate Format

[0213] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0214] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then.re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0215] Oligonucleotide Analysis—96 Well Plate Format

[0216] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0217] Cell Culture and Oligonucleotide Treatment

[0218] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 4 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR.

[0219] T-24 Cells:

[0220] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum A (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0221] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. A549 cells:

[0222] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0223] NHDF Cells:

[0224] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0225] HEK Cells:

[0226] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0227] Treatment with Antisense Compounds:

[0228] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0229] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transefection experiments.

Example 10

[0230] Analysis of Oligonucleotide Inhibition of Phospholipase A2, Group VI (Ca2+-Independent) Expression

[0231] Antisense modulation of Phospholipase A2, group VI (Ca2+-independent) expression can be assayed in a variety of ways known in the art. For example, Phospholipase A2, group VI (Ca2+-independent) mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0232] Protein levels of Phospholipase A2, group VI (Ca2+-independent) can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Phospholipase A2, group VI (Ca2+-independent) can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0233] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0234] Poly(A)+mRNA Isolation

[0235] Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0236] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0237] Total RNA Isolation

[0238] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RWl was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

[0239] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0240] Real-time Quantitative PCR Analysis of Phospholipase A2, Group VI (Ca2+-Independent) mRNA Levels

[0241] Quantitation of Phospholipase A2, group VI (Ca2+-independent) mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.

[0242] With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0243] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art. PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out. 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0244] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.

[0245] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0246] Probes and primers to human Phospholipase A2, group VI (Ca2+-independent) were designed to hybridize to a human Phospholipase A2, group VI (Ca2+-independent) sequence, using published sequence information (GenBank accession number AL022322_COMP_TRUNC, incorporated herein as SEQ ID NO:3). For human Phospholipase A2, group VI (Ca2+-independent) the PCR primers were:

[0247] forward primer: GGCGTCACCAACTTGTTCTCTAA (SEQ ID NO: 4)

[0248] reverse primer: CGGTCACTCGAGGTGTAGTCG (SEQ ID NO: 5) and the PCR

[0249] probe was: FAM-CATTCCGGGTGAAGGAGGTGGCT-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were:

[0250] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

[0251] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

[0252] Northern Blot Analysis of Phospholipase A2, Group VI (Ca2+-Independent) mRNA Levels

[0253] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0254] To detect human Phospholipase A2, group VI (Ca2+-independent), a human Phospholipase A2, group VI (Ca2+-independent) specific probe was prepared by PCR using the forward primer GGCGTCACCAACTTGTTCTCTAA (SEQ ID NO: 4) and the reverse primer CGGTCACTCGAGGTGTAGTCG (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0255] Antisense Inhibition of Human Phospholipase A2, Group VI (Ca2+-Independent) Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0256] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Phospholipase A2, group VI (Ca2+-independent) RNA, using published sequences (the complement of residues 1-70000 of GenBank accession number AL022322, incorporated herein as SEQ ID NO: 3, GenBank accession number AF064594, incorporated herein as SEQ ID NO: 10, and the complement of residues 40601-42896 of GenBank accession number AL021977, incorporated herein as SEQ ID NO: 11). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2¹-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analized for their effect on human Phospholipase A2, group VI (Ca2+-independent) mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Phospholipase A2, group VI (Ca2+-independent) mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ISIS SEQ ID TARGET % ID # REGION NO SITE SEQUENCE INHIB NO 129845 Intron 3 7118 aaaaccaagcttgcctgctg 81 12 129846 Intron 3 11373 ggtgtagtcggccacagcca 70 13 129847 Intron 3 15023 cctggctaagagtagatggt 40 14 129848 Intron 3 16574 agtatctatgctatcatagt 39 15 129849 Intron 3 27859 gctcaatcacaaattgcaaa 65 16 129850 Intron 3 31022 atacctgtaatcccaccact 52 17 129851 Intron 3 32898 tggtctcccaaagtgctggg 49 18 129852 Intron 3 69954 gggaaggcatcctgtgccgg 50 19 129853 5′ UTR 10 61 acacgaggaatatccaaagg 79 20 129854 5′ UTR 10 73 tcagaatcggagacacgagg 79 21 129855 Start 10 128 aagaactgcatcttctgcgg 86 22 Codon 129856 Coding 10 194 tccttcacccggaatgggtt 77 23 129857 Coding 10 322 atccactctgtgagttcctg 70 24 129858 Coding 10 336 ctggaagagtcggaatccac 61 25 129859 Coding 10 376 actgatggaaattcactagg 76 26 129860 Coding 10 458 atgaggtcggtcaggtgctg 47 27 129861 Coding 10 533 cggctgtgatggaagcactc 54 28 129862 Coding 10 608 tcacccttgcggcaggccag 65 29 129863 Coding 10 632 accagctccaccaggatctc 92 30 129864 Coding 10 680 gtctctcccttgtagtcggt 70 31 129865 Coding 10 701 tggacagcataatggaagac 73 32 129866 Coding 10 958 tgtggatctggctgctgtcc 5 33 129867 Coding 10 1009 ctgcgttcttggcccagtgg 54 34 129868 Coding 10 1026 cagcatgcgggccatctctg 51 35 129869 Coding 10 1052 ctgttcacgttgcagccccg 73 36 129870 Coding 10 1063 cggagctggtgctgttcacg 78 37 129871 Coding 10 1082 tgcagggccgtgttccccgc 63 38 129872 Coding 10 1196 ttcgacatggccaggtgcag 70 39 129873 Coding 10 1304 agtctgccgattttggaggc 68 40 129874 Coding 10 1313 ctggtgacaagtctgccgat 64 41 129875 Coding 10 1435 gagctctttccagggagaag 59 42 129876 Coding 10 1485 gtgcatgagatcctgtagtt 46 43 129877 Coding 10 1541 cgcttctcgtccctcatgga 66 44 129878 Coding 10 1557 caggtggtcgtgggtccgct 15 45 129879 Coding 10 1601 tggatgatgatgaggccttt 0 46 129880 Coding 10 1627 aggccttctcgatggcgatg 42 47 129881 Coding 10 1701 aatggccagggccaggatgc 52 48 129882 Coding 10 1739 tacatgccgcgcatgtaggc 56 49 129883 Coding 10 1763 aacacctcatccttcatgcg 45 50 129884 Coding 10 1832 gtgtgctccccaaactcccg 8 51 129885 Coding 10 1835 ttggtgtgctccccaaactc 77 52 129886 Coding 10 1869 cagcatcaccttgggtttcc 28 53 129887 Coding 10 1871 gtcagcatcaccttgggttt 43 54 129888 Coding 10 1881 cagtgtccctgtcagcatca 60 55 129889 Coding 10 1885 cagacagtgtccctgtcagc 70 56 129890 Coding 10 1894 gctgccggtcagacagtgtc 46 57 129891 Coding 10 1932 tggagcatcgtagttccgga 41 58 129892 Coding 10 2006 accagctggtctgagggctg 16 59 129893 Coding 10 2045 taagtaggagctgccccgct 80 60 129894 Coding 10 2056 tgggtcggaagtaagtagga 41 61 129895 Coding 10 2096 gggttgttggccagcagccc 21 62 129896 Coding 10 2129 tactcatggatctcggtcat 74 63 129897 Coding 10 2138 tcctgattgtactcatggat 59 64 129898 Coding 10 2198 cccagggagacaacgatgga 44 65 129899 Coding 10 2249 ggacggaagacatccacaca 56 66 129900 Coding 10 2280 aacagtcttggccagctccc 79 67 129901 Coding 10 2306 atcttgcccagttccttggc 71 68 129902 Coding 10 2320 aacagtccaccaccatcttg 2 69 129903 Coding 10 2329 gatccgtgcaacagtccacc 76 70 129904 Coding 10 2331 tggatccgtgcaacagtcca 22 71 129905 Coding 10 2379 gccgaccatctcgcaccagg 20 72 129906 Coding 10 2389 agtactggatgccgaccatc 53 73 129907 Coding 10 2394 tctgaagtactggatgccga 39 74 129908 Coding 10 2439 actgacctcatccagcatga 68 75 129909 Coding 10 2475 ctcggtctcccagagggcgt 72 76 129910 Coding 10 2485 agatgtagacctcggtctcc 51 77 129911 Coding 10 2490 ctcatagatgtagacctcgg 8 78 129912 Coding 10 2495 cggtgctcatagatgtagac 59 79 129913 Stop 10 2544 ggaccctcagggtgagagca 53 80 Codon 129914 3′ UTR 10 2663 caggcctggtctatggactc 20 81 129915 3′ UTR 10 2695 accagcctcgggcaggcagc 72 82 129916 3′ UTR 10 2944 cacccaccaggaagcctggg 0 83 129917 3′ UTR 10 3000 catttctttagtcccagccc 67 84 129918 3′ UTR 10 3038 cggcctgggctttcccagct 32 85 129919 3′ UTR 10 3050 atcccactcctgcggcctgg 16 86 129920 3′ UTR 10 3135 caggagtgacctttgagagc 58 87 129921 3′ UTR 10 3200 ttgaatccaaatgatttatt 46 88 129922 Intron 11 2194 tactggttataaagctttac 90 89

[0257] As shown in Table 1, SEQ ID NOs 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 60, 61, 63, 64, 65, 66, 67, 68, 70, 73, 75, 76, 77, 79, 80, 82, 84, 87, 88 and 89 demonstrated at least 40% inhibition of human Phospholipase A2, group VI (Ca2+-independent) expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 16

[0258] Western Blot Analysis of Phospholipase A2, Group VI (Ca2+-Independent) Protein Levels

[0259] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Phospholipase A2, group VI (Ca2+-independent) is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 89 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 atgcattctg cccccaagga 20 3 70000 DNA Homo sapiens 3 tataaccagt atggccctgt acaaatacat gattttactg ttgggtcata ggcagaattg 60 aaaggagtgg aaagggaaag ggctagaatg agaagacaag gagagaagga tgagcaggag 120 gtcccttgtc tttcagtgta tcatttggca gtgtttgttc atcacctctt gtgagtcagg 180 ctcttctgta tatgttctct tcaccattca ttcagtcatc gtttatctaa caaatattta 240 cggactgtct gatgttctag gctacccttg gtgtggccaa tgatattctc acttcacatg 300 agaaaaagag ggcgcatgag ttgcttgagg tcacaccgat gaggttgaag ctggaatctg 360 acaccagagc ctgtgtgatt gtcctgatgt gtaaggtttg ctactgttta ggacacttct 420 tgttccttcc gtggattact ttcttgttac atgagagact ccccaaaagg gagtttccct 480 cttactcagc agaatgacat cactaagaga tacagaaagg agaaatttaa atactctgtg 540 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtc tgcagacatc tattgggtgt 600 gtgtgctggg ggaagtaggt aggcaaggag taaggggccg tagtgaagag tgtgttacaa 660 acttcacttt gtagagggtg gctccctggg agaatatgtt ctgtgtcttt cacagtggat 720 tttgataggg tcatgtctta aatgcctctt ctcgttttcg gatggtgatc tcatccagtc 780 ttatttcttt aagtgccgtc tctattctgt cagcccacaa atttatatcc ccagtccagt 840 ccagaccttt ccctggaatg acatactcaa atctagttgc ttactcagca cctcaaactt 900 aggcttttca aacttgaagt atttatttat tgatttattt attttttgag acagagtctc 960 actctgtcac ccaggctgga atgcaatggc acgatctcgg ctcactgcaa cctccacctc 1020 ctgattctcc tgcctcagcc tcccaagtag ctgggattac aagcatgcgc catcatgccc 1080 aactaatttt tgtatttttg tagaggcggg gtttcaccgt gttggccagg ctggtctcga 1140 actcctgatc tcaagtgatc cacctgcctt ggcctcccaa agtggtggga ttacaggtgt 1200 gagccaccgc gccaggcctc aaacttaaga gtttagaatt gagctcctgt tagttctgcc 1260 ctccatccct gaccccactc ttcaccaaaa catgtccctc ccactctctt tctcatctta 1320 ggacatagga gtattgttca ttcagttgct caggtcaaag ctgtgagatt atctatgatg 1380 cctctctgtt tcttacccca cacccagcca gagtgatctt gttaaaatgt tgtttctttg 1440 cagtggcctc tcctctcatg gcacatgcct ggccccctgt tgcctctctg atgtcactgg 1500 cttccttgct tttcctggaa ctcgctaaac atgttccagc cccatggcct ttgcacacgc 1560 tctctttgcc tagctattca tatgactttc atctctctcc atcaggtatt tatttaagat 1620 caatttcttg gtgaagactt ctctcctcac ccaatctaaa atttcactct ccacctcagc 1680 acttcctatc cccattctct ggtttttctc ctcagtagtt aattgtcatc taaaaccttt 1740 gtaaatattt acctgtttat cttctttatt attagtagct ctgtctaaaa tataagttcc 1800 aggatgtcag ggattttgtg gggttttttt tggttggttg gttgttttgg ttttgagaca 1860 gggtctcact ctgttgccca ggctggagcg cagtggtgcg atctcagctc actgcagcct 1920 ccgcctcctg ggtttgagtg attctcccac ctcagtctcc caggaagctg gaattacagg 1980 tgcacgccac cacacccagc taatttttgt attttttggt agagatgggg tttcactatg 2040 ttggccaggc cggtcttgaa ctcttgacct caagtgatcc acccgcctcg gcctcccaaa 2100 gttctgggat tactggtgtg agccaccgcg cccggccttg ttttgtttta agacagggtc 2160 tcactgtcgc tcaggctgga ttgcagtggc acaatcacag ctcactgcag cctcgacctg 2220 ctagacttaa gcaatccccc cacctcagcc accttagtag ctgggactta caggcatgag 2280 agataccatg cctgggtaat tgttaagttt ttgtagaggt gaggtctcac tatattaccc 2340 aggctggtct caaagtcctg agcttaagtg atcctccttc cttggcctcc taaagtgctg 2400 ggattacagg agtgaactgc cgtgcctgac cagtctgttt tgtttacgat tgtatcctca 2460 gcggttagaa caatgcctgc cacaatccat ctcagtaaat atttgtggag tgagtcataa 2520 catgggccac actgtaccgt gccaccagcc caaagatctc ttaaagtcac ccctgtctca 2580 tgtgcttggg agctccagtg acagtttcgg ggcagtttgc ttccagctgt ggtccagcca 2640 agcggagtga atgaaagaga tgctttttcc ttttgtgaaa ggacgttttg ctctggctca 2700 cgtgagcagc agtgcatttt gagacgaggg ccttggcttt tggaatctaa gagcctgttg 2760 agcaagcctc gctcagccac cttctttcag ctttctcctg aaggcagttt gaggattaag 2820 agcctgatgc tctggattcc acctaattgg gttcagatcc tggcaaactg tgtaacctgt 2880 gccccagttt cctgtgtaat gtggagagac ctggaccttt tcttacaggt gtttggaggg 2940 ttaaatgtgg caattcggcc gggcgcagtg gctcatgcct gtaaccccag cactttggga 3000 ggtggaggtg ggcggatcac cggaggtcag gagttcaaga ccagcctggt taacatggtg 3060 aaaccccgtt tctactaaaa atacaaaaaa ttagccgagt gtggtggtgc gcgcctgtaa 3120 tcccagctag tctggagact gaggcaggag aatcgcttga acccgggagg cagaggttgc 3180 agtgagccaa gatcacgcca ttgcactcca gcttgggcaa caagagtgaa actctgtctc 3240 aaaaaaaaaa aaaaaaaaaa agtggcaatt tacccgaaac tgtggtgaag cgtctggcgc 3300 cctgtgggtg ctgggcagca gtgggtgctg atggggtgtg aggggatggg atcaagccct 3360 ttcccactaa cttaggaaag aggttggaat gagctggggt cagagggaac atttgcgggg 3420 acattttgcc gggaggctgc cttggaggtt cagaggctca gatgacccct gtgttcattc 3480 agtgttttct cacccacaga ttgagcacct gctgtgtggc cagccctttg cctagtgctg 3540 ttcaagagtc agaggacata ggcaggcaat cccccaccac aacctgccac cccatttcct 3600 ctcttggggc cttgatttct ccacatgcca aacagggaga atgattgcac cttccttgtt 3660 tctttttttt tttttttgag acagtctcac tttgtcatcc aagctttagt gcagtggcat 3720 gatctcggct cactgcagcc tctgcctctc gggttcaaag attctcctgc ttcagcctcc 3780 caagtagctg tgattatagg cacccaccat cacgccaggc taattttttt tgtagtttta 3840 gtagagatgg ggtttttttc tcacacgttg gccaggctgg tcttgaactt ctgacctcag 3900 gtgatctgcc catctcaccc tcccaaagtg ctgggattac aggtgtgagc caccgcgccc 3960 ggctgcgcct tccctgtttc tttgtgagct tggaggtgga tcacaagtgt gaaacccttt 4020 gccaaccgtg aggcatggtg gccttgtgag aggacggagg ccggtgtgtg gtggcatgct 4080 cccgtgctgt gtggcacaga cggagctaca ggaaggaagt tggggcagct tggcactctg 4140 agtggaccct gagtttccag cctgctctgt cttgactggg tgggaggagg gtgctcagga 4200 gggtgtcact gctgccaggt tctgctgcca ggaccatcca acagttgttc caatcatggg 4260 acatcctgtc gtcagcagct ggggctcgtg aggcctgatt tccacctcgg gcagttctgt 4320 gtgtgctcct ggaatccccc acgtggccaa gcctgtgagg tctgcttcct ggcctgctgc 4380 ctgtgggttc ctgtcacgca cctctcgggc agctgtccgg ctcctctgct ctcggcttag 4440 tcaagaagga agcagaaggc ttggagtctt tcctgcttgg actaggagtg agctcagcct 4500 aagtgccctg actatatata aagctgaact ttccagaccg aagcctgggg atggatcagt 4560 ccgatcactt agggccgctc ctgaaggagt gcaggtgcta gatacaggtt ctcccggtgg 4620 aggagcgaac cccttgtggg ccgttagcta ctggtgggat ttgagcatgg tatttcccta 4680 tccgttgtgg agaggagcat ggcagctcag agtgggtggg aacagtgtgt ggtccctgtt 4740 atcaccttca agggaataaa gtgtgtctgg gggcttcaga ggtgctggcc ccatgggctt 4800 ataggtggtt cattcattca ttctcattca ttcattcacc aaacttatta aacacccact 4860 gtgagtcagg aacatcatgc caggtgctta gggaaacaaa tcaaagcggg ttgggtgagc 4920 agcccaagcc aacagatgca cccccattct cacatggtgt gtgttcctgt gggctgggcc 4980 ctgggtggtg tggggtgctg gggagaagag gaaggcacac caggctgcag ggcggaggtg 5040 cgggtatgaa ttttcagagg agatggcacc tgagctgtat cttcatggat aagaagagat 5100 ttgttcattt gtcctttgtg gagcacacat tcaggttaag aaagtgggga ggatgggcta 5160 cgctgatgga gcgtatgtgt ggtatcccca agtgcactga gacacgagtt tgacgtgttc 5220 tgggaagatc gatggtgcgg cgtggctgga gtagaggtga gggatgagtt ggaggggtga 5280 gttgggccag ctctcatgga tgctggagtc tgggctttcc ctgggaggtc atggggcgcc 5340 actggccagg gtcagcaggg agacctgcag ctggatttgc atcgtacagt gggcatggag 5400 gggtgtcagg ctgggttggg aaccgtgcag gctggaaggg cactctggtg ctctgagcat 5460 tcagaggggg cagctgggcc ttgaaggatg agcaggggct ccagcaggaa ctggagagcg 5520 cattccaggg aggacgcaag tgcaggcccc tttcactaca gaaaagcagt tttcacacag 5580 accttccaaa ggggaggaga ggggactgcc tgggcctggc agctgcggca ggtagcagca 5640 atcagagggc agggaccgct cctcccgtgc ctgagacctg cccagggtcc ttcagggaat 5700 tcgtagctga ggaggaacca gatttttggg actgagttct ccctcaccac ccgctctgag 5760 ccaccctctt ggtcccttgt cctctactgc acttcttccg gggcttggct cctcgtcatg 5820 agaggagctt ccctgggcat ggcgaggggc gtgactgaca tagcagccct ggacaagtgg 5880 ctccaggtga cctgtgttca cgttgatcac agcactaacc acggcccctc ctctcagcac 5940 cttcccctct cgagccgtcc agtcccagtg aaaatgctca ccgttcatgg gtgggctgag 6000 caagcccctg atctctgcct tcctccgtgt tctgatctgc ttcatctgct gtgaaggcct 6060 ctcgccattt cccccgctgg caaactcctg ttcttcctgg aaagttcagc taaaaagtca 6120 tcccctttag gggtccttcc ctcaggggac cgtgcacttg gcgagacagg gactggtctt 6180 gggtctctct gttgactgtt ttatgaaccc aggtagtcac ttccagggaa ctcatcagag 6240 atgctaccat tggctccaat ctccctctgt tcccactcga ggttttttgt ttcttcgttt 6300 gtttttcttt ctttcttttt ttttgagaca gagtctcgct ctcgcccagg ctggagtgca 6360 gtgacatgat ctcggctcac tgcaagctcc gcctcccggg ttcacgccat tctcctgcct 6420 cagcctcccg aatagctggg actacaggca cccgccacca cgcccagcta attttttttg 6480 tatttttagt agagacgggg tttcaccatg ttagccagga tggtctcaat ctcctgattt 6540 cgtgatccgc ccgccttaac ctcccaaagt gctgggatta caggcgtgag ccactgcgcc 6600 cagcccgttt gtttttgttt tttggtggac atcaacttta atttgccctt tgttttgttt 6660 tgtttttgag gcagggtctt gctctgtctc ccaggctgga gtgcagtagt gcaatcttgg 6720 ctcactgcag tctcgacctc aagaggatca agcaatcctc ttgccttagc ctcccgagta 6780 gctgggacca caggcgcaca ccaccatgcc cagctaattt tattttatat tttgtagaga 6840 cagggttttg ccatgttccc caggctggtc tcaaattcct gggctcaagc aatccgtctg 6900 cctcagcctc ccaaaatgct ggaattacag gcacgagcca ctgtgcccgg ccttaatttg 6960 ccctttgaac atgtacaatt cagggcctct ctgggcctca gtttcttgat ctctgagatg 7020 ggaagatggg ataataatac ttgttcctcc cgtccttcag ctcctccagg ggaccctcac 7080 accagcacac tctctgtcag tcttccctca atgaacccag caggcaagct tggttttgac 7140 agaggatggg tgagcagaga taaggaaaac ggccagaaat atctggcttc cctcagcagg 7200 ggcaccaaaa ttggtacctg gaaattgcct ggcttggaca ggcagaattg tcactgtcaa 7260 cccccaccac caaaggtctt gggtctttgt ctttctgctt gtggtgtttg agagagctgc 7320 ttgacacagc cgaggtgcgg tctgtgtcag gtgacctggg catcctgggg gtgcagggag 7380 aagcccaggt aaggaggagg aggggccggt catcagtggg gaaaagtgat tgtctacccc 7440 acgggaaggt caagggtagg acgctgtgag aagccagggg gagagatgaa gtgaggcatg 7500 atgcgggggg ccctcatcca ccatccccct cttgcagctg tgctgggaag agcctaggct 7560 ccagggtcca aggtggcagg gacagaagga agcacgtgtg agcactttct ccctgctggc 7620 ccgccttcct ggttggcaat gccgtgagtg ttccaggatg tggccccgtg gctgtcattt 7680 cctttcttcc acctagtgat aacaatggcc actgtttgtg tgcccacagt cggcctgtgt 7740 cccagacacc ttccctccat tgcgtcatct cgtcctcctg cctaccctga aggctggatg 7800 ccagccaagc ctgtcgtttc tgtggccatg ctgtcgtggg tgtgtggggg gcatccccta 7860 gccccaggct ctggtggggg gtacacaggc tgggccattg acgaaggggt gtgtggaatc 7920 tttctttgat ttcactgcac ccctggtgac tttctccctc aatctgaggc tcatgggtgc 7980 tggatgctct tgactctctg cccttcctgg gcgcacccct ccgcgtccct gtgggcccgt 8040 cctcggggta gcctctctgc gctcttccag gccaggcctg agtcccacag gtccccatct 8100 tcccggctgc agcccccaga ggctcagcct atactgctac ttcctttgaa aactataatc 8160 gctttacata gcatctggaa aatagggaaa aggaaaaaac aaaagcccat aaacccagca 8220 ccctcatcta ctcaatggca tgttttctgt cagtctatct tggtttgttt tctatcaggc 8280 tttgtttttt ctatgcataa tatttactca gttaaatttt tgttttgaca aactttctgt 8340 gagccaagct tgtggacgga gatgaaaaga cgagcagctg cctttgagaa actcagactc 8400 tgggccagcc catctgggtg ccagctttca ctgctccacc cactagctgg gtgaccctgg 8460 gcccgtgact ttacccctct gttctttggt gttctcatat gtaaaatgag gataataata 8520 gtaaccagtt catagagtgg ttgtcagaca gaatgattaa tgtacgtaaa gctttcagga 8580 tggtgccagc aagatgcaca ggagtgtttg gtaggaggat gacgaagctt accgtctggc 8640 aggaggggca gttgtaacat gctgggctgt gatagtacag atgtgtttaa agtgagggag 8700 agcccagagc aaggagagag aacctggcat ctggtgatgg tgtatcaaga acattttagg 8760 ccagacacgg tgactcacac ctgtaatccc agcactttga gaggctgagg caggcagatc 8820 gcttgaaccc gggcgttcga gaccagcctg agcaacgtgg taagaccctg tctttataat 8880 tttttttttt ttttttctga gacggagtct tcactctgtc acctaggctg gagtgcggtg 8940 gtgctatctt ggctcactgc aacctccacc tcccgggctc aagtgattct cctgcctcag 9000 tctcccaagt agctgggatt ataggtgccc accaccatgc ccagctaatt tttttatatt 9060 tttagtagag acggagtttc accatgttgg tcaggctggt ctcaaattcc tgacctcaag 9120 tgatctgccc tcctcagcct cccagagtgc tgggattacg ggcatgagcc accatgcctg 9180 gcctctataa aaattgtttg gaagaaatta cctaggcctg gtgatacaca cctgtagtcc 9240 tagctactca ggaggctgag gtggaaggag gtagaggctg cagtgagcca tgattgagcc 9300 actgtactcc agcctgggca acagagccag accctgtttc aaaacaacaa caacaaaaac 9360 atttttaagg aggaggctga gaaccacgag aaccaagctt ggatttgaag aaggatcagg 9420 gctttctcag acagaaaagg agaggaagaa cattccagat agacatcatt gagagatggc 9480 actgagcctt gtaatgggac actgcgaact tttcttcagt tgttatgatc tttttttttt 9540 tttttttgag acggagtttc gctcttgttg cccaggctgg agtgcaatgg cgccatctca 9600 gctcaccgca acctccgcct cccaggttca agcgattctt ctgcctcagc ctcccaagta 9660 gctgggatta caggcatgcg ccaccatgcc tggctaattt tttgtatttt tagtagagac 9720 ggggtttctc catgttggtc aggctggtct caaactcctg acctcaggtg atccgcccga 9780 gtcggcctcc caaagtgctg ggattacagg cgtgaaccac cgtgccttgt tggtataatt 9840 attagtattg atcttttttc cttttcatgt gcctcttagt gggccccttg cttttgtctt 9900 ccttctgtcc ccagggttga tcctgtgctc agcacatagg ccagggaaga atgtggctgt 9960 gacccagtgt ccactcagat gtcctctctc ccccagggca cccatactct gtccttagtg 10020 tcttttgtct ggtgacatct tggcacagag gcagagggcg gcctccgtct gattggcgcc 10080 tggctttggt gacagtacct taggaaggca gtgtattttc tgggaaccta ggaaataaca 10140 aggtatctaa ttccatcatc aacactgggg acaatttcca tggcagaaac tatgactctg 10200 aatatgcagc gtaaagttac cacccagaca ctgctgctcc tgagtctcga atggcatctt 10260 ctgcaggggc tcccccacct gcccctgcag aaggtgcagt tcgttcattt ccaaggagct 10320 tgcttttgtg tgctgtctct cggagctctt tgggaaacct gtggtagcag gaaacaaagg 10380 atttagacca atgccgatga tccccttgct ccgatagagt gtgacatgat gctggtcacc 10440 ttttggtatc taggaggggg gacattgatt ttgtttgcgg aagtgaaatg atccatgctg 10500 gcctcttttt tttttttttt ttttgagaca gagtcaccct atcgcccagg ctggagtgca 10560 gtggcgcgat ctcagctcac tgcaacctct gcctcccggg ttcaagtgat tctcctgcct 10620 cagcttcccg ggcagctggg actacaggcg cctgccacca catctggcta atttttgtat 10680 ttttagtaga ggcagggttt cgccatgttg gccaggctgg tctcgaattt ctgacctcaa 10740 atgatccgcc cgcctcagcc tctcgaagtg ctgggattac atgcctgagc cactgtgtct 10800 ggtccatact agcctctttg agtgctgaaa ggttatctct gtaacttatt tagtgagaac 10860 cctccatttg cccagggcat tactgcctat aatgtatgtt cacatgtgct atctcctttg 10920 gtcaatacca cacactacag gtgaggaaac tgagtctctg ctgggacact gggtcaggaa 10980 gcaggcagtt aaggtcacct gtgcacagtg gcagcacagg gggacagcct tctaggtagc 11040 cagcctggcc tgagggttct gtcactttta cctcccactc agtgttgttt ctctttcttt 11100 cccctccttt taagatccct ccaggcccag gatctgggga caacgctggt ggggagtgat 11160 ctgggtgtct gtgcaggaaa ccgctgcact gggtgagatg ggcatgggag actgtgcctg 11220 gctgaccaga gacctcgttt tcttccagac agagggggaa gacggtgggg cctccccacc 11280 tgccccgcag aagatgcagt tctttggccg cctggtcaat accttcagtg gcgtcaccaa 11340 cttgttctct aacccattcc gggtgaagga ggtggctgtg gccgactaca cctcgagtga 11400 ccgagttcgg gaggaagggc agctgattct gttccagaac actcccaacc gcacctggga 11460 ctgcgtcctg gtcaacccca ggaactcaca gagtggattc cggtgggtga tgctcagcgg 11520 gaacatactt ctctcctttc actcccccac gtctcggatt ggaggtctta ttggccgaga 11580 agcagtttct ttgagtctct gtctgagggg tctggagagg ggtgggagag aggggagact 11640 gtactcttgg accagaggga agggcttccg agactccaca cttctggccc atcagtgtcc 11700 cagcacgagg ctgaggatgg aagcctgggt tgtttctggc ctggaggctc gacctatatg 11760 gacaaaggga agaaggaagg acagggagac tgagagtgtc cacatcagat aaggcccatg 11820 gggagaacct ttagccctgg ctctttattt ttggttttgg tttttgtttt ttgagacagt 11880 cttgctctgt tgcccaggct ggagtgcagt ggcgcaatct cggctcactg caacctctgc 11940 ctcccaggtt caagcgattc tcctgcctca gcctcctgag tagctgggat tacaggcacc 12000 caccaccacg cccagctgat ttttgtattt ttagcagaga cggggtttca tcatattggc 12060 caggctggtc tcaaactcct gacctcaggt gatctgccca ccttggcctc ccaaagtgct 12120 gggattgcag gcgtgagcca ctgcacctgg cctagccctg gctctttaag ggcatggccg 12180 ttctcatagg ggctgcccag ggtttgctta ggaaggctgg acagagctgt tgcctcttgg 12240 ggcatgctgg gactcccctg ggcctgaaga gaaggctcaa aagccaggac aggcctgagg 12300 gaaccgggcc aacggcagag ctggttggga aaaggggaga agcagccaag gttccaaatg 12360 caggccccag cagtgcttgg cagactggca tctagttggg ctcagacaga tatccctgtt 12420 tccgctcacc tatccatctg gctgtcctgt cagccatcca ttcaacatgc accaagcacc 12480 tccagtgtct ccagagtcct gggcttcggg ccagtcctgg ctggcctgtg agatgacaaa 12540 catgtcctgt gtctttaagg aacgtacagt ctggtggggg aggtccggtg caaacaggcg 12600 tcatagcacc ccttgaggag tgctgtgaca gagggagagc ctaggaaaca gtgtggatca 12660 caaggaggtg attgagagct actctgcgtc tggaagagtg aggaggagcc tgtgtggcag 12720 aacaaaatgg ataaggtcgt tccagccgtg cagaagtgtg gaggtgtgag cctgcaggca 12780 ggtggctctg gttggatccc aggtgcatgc agggctttgg tgagagtgag gctgaaggaa 12840 gcagctgggg actcattctg actaaagagg catggcagga ggggtctctg aaagtgtcag 12900 gctgggggcc aggggctgcc accaacagaa gagcaaggca ggtatcaggg ccccatccaa 12960 tcaggtgaga attcagagcc tggctccaca aggaacctga ggcagaggca cccacagcag 13020 gtgcttgggc taaatgcagc tcagtggcca gtgcaggcac cagttttaat acagtggggc 13080 cattccagat tctgagtgca gatggggctg agcagaccgc cttccagctg gactcgatca 13140 ggactgaccc tggacggggc aggcttgcac ttgtagcttc agagctccac tgcaggcaca 13200 gctgaccagg gcagcgggtc cactccttgg agagctggac agccccactt ttgaggtaca 13260 cggccctggg gataccacta atctccacgt accaataatc accaagttct gtcagttgta 13320 aaatcccttg acaggattcc agatttgtct cagccagctt aagccccagt gtgggcagac 13380 ctccatccca ggaggattta gtcctttgtc agcagcttct tagctacacg taagctattc 13440 tgtggtactt tatagtttac aggatttttt gttttctttt tttttttttt ttgagacaga 13500 gtctcgctct gtcacccagg ctggagtaca gtgacgcgat ctcggcttac tgcaagctcc 13560 gcctcccagg ttcacgccat tctcctgcct cagcctccca agtagctggg actacaggca 13620 cccgccacca cgcctggcta atattttgta tttttagtag agatggggtt tcaccatgtt 13680 agccaggatg gtctcaatct cctgacctcg tgatccgcct gcctcggcct cccaaagtgc 13740 tgggattaca attttacagg atgtttttaa gtatgttggg ttagcacatt ctctgccaat 13800 ccgtggaggt gggcaaggaa ggcagtattc cattttatgc atgagaaaat agccttggga 13860 tgataagtga ttcatcccac ttatacacag gaagcagtga agaaacagaa tcagtgtctt 13920 ctggttccca attcatttct ctgtgctgct gtgttgtttc catgtatgta tgtatgtatt 13980 tatttttttg agacgttaag tcttgctctg ttgcccaggc tggagtgcag tggcatgatc 14040 ttggcttaca gcaacctctg cctcctgggt tcaagctatt cttctgcctc agcctcccga 14100 gtagctgaga ctacaggtgc gtgccaccat acctggctaa tttttgtgtt ttttagtaga 14160 gacagggttt cactgtgtta gccaggatgg tcttgaactc ctgacctcgt gatctgcctg 14220 ccttggcctc ccaaagtgct gggattacag gcatgagtca ccatgcccgg ctttgtttgt 14280 cttatttcta aattttattg atttatttat tttcacagtc atagctctgg agtctttttt 14340 atcttctccc ctctaacctg ctcccacccg aacctttcca tttcagcaga tagcatcatt 14400 gcaacgggct gtgccagatc cagccatcac atccaccttt ccagccaaag ggagggtgga 14460 agggcaggat gcagaaggtc aatctccccc tcctcaggga catttcatgg aagttgcacg 14520 cattggccag aacccggtca catggccaca gctaactgca aaggatgctg ggaagtgttg 14580 cctgtgttgc agacagccac gtgccagtcg aaaatcagag gttctttctc cctccgtctt 14640 gctgctggct cgcttctcca gaccacagtc attgtgcacg tgtctcctgc tgctccggct 14700 gtggcctgca ctcctctcat cctcttccaa gctgctgtca cagcgctgtt tccagagggc 14760 aaatctgatt ggtcactccc ctgctttact ccccaagggc ttcctgtttt cttttatttt 14820 tatttttatt ttcatttttt gagatggagt ctcgctctgt cgctcaggct ggagtgcagt 14880 ggcacaatgt cagctcactg caagatctgc ctcctgggtt catgccattc tcctgcctca 14940 gcctcccaag tagctgggac tacaggcgcc cgccaccacg cccagttaat tttttttgta 15000 tttttagtag agacggggtt tcaccatcta ctcttagcca ggatggtctc gatctcctga 15060 ccttgtgatc tgcccgcctc agcctcccaa agtgctggga ttacaggagt gagccaccgc 15120 gcccggccag cttcctgttt tcttaacaca tcaggtgctt aatgacttaa aagggcccag 15180 tgtgatcaag tttctgcctc tgtgttctca gctcatgccg ccttgtgctc ctggcctttg 15240 cccaggctct tgctgtggcc tggaatactc ccctttcttt gcctgccttc cttcctcttg 15300 ccctcccctg acctgcaggc tggattagga gtttctgctg ggcatcccat ggcacccgtc 15360 cctgcactgg ggatcctgct ttcctggtag gccacagacc ttgtgagggg agggactgtg 15420 tctgttgggt tcatgacagt gtgcccagcc cccagcaggt gcctggcgta gatcaggttt 15480 gtggaacaaa tgaatgtgat atattaattt agctttagag tattcattga gggctgctgt 15540 gtgccaggcc ctctgtgcca cctgggtgga gggtggagga ctgttgcatt gagggcagtt 15600 gaaggagtga gctgggaagt gggaggtgga ggatggggac tgggatgcag ggtttgaggt 15660 tatcaggggt cccagaagca gtaatgccaa ggcctaggct gtgacctgga gtgagggctg 15720 cgatggggtg gggtggggtc gaggctgtgg aggagggtca tcagggcact gctggagccc 15780 tggggttctc cagggccggg ggtggtcatt gcagtgttct cccagctcct ctcaacaccc 15840 tgcggtctgt ggagagcagg cctggtgcac tggacttgcc tctggccagg gctgagtttc 15900 tgccttctaa acgatcttcc tcctgtgttg ccctagaggc ctctggactg tcctgagtac 15960 ttgacgcgtg ttagagtgag catgggctgc tagccttccg ctccaggtgg aggagtttct 16020 gttgttcttt tgctgctgtt gctgcagtga gctgccaggc tgagcttctg gatggacctg 16080 ggggatggaa ttaggagctt ctgaattctt gggttgtata acctaaggcc ggagagtccc 16140 acagcactgc tgctgggcgg agagactgga ccctgctgat acagacagcg acagcagcag 16200 gagcatctgc agtttcccag caggtcctgc atgcctgggc ttggcactag tctaagtagt 16260 aactcatgtt atcctcacga cagtcccacg aggtggggtc attactgacc tcctattaca 16320 gatgaggaaa cagaggcttg cagaatttga gatcacacag ctggtcagcg tagcgattat 16380 tcagaccctg tcacctggct ccagaaccca ctcgtttacc tttcttacat cagggctgct 16440 ttgtagtgtg gcgagctgag tggagctcca cccgcctggg ctgatcctgg cccttcctgc 16500 ctgacctatg gtgcgggggc cctggctctc cccgtgtgag cccccagggg tcacttgtaa 16560 tatgcctgtg tacactatga tagcatagat acttctgttc ctggtacaga ttaatcattt 16620 acggctggca ttgacttgta acagatgaca tttgatgaaa tcaaccagcc tctcactggt 16680 atctcagcag aaacagggct gtacctccca ggcatcctgg gaaggatgat agaagtgtgt 16740 gatctgtgct tccctaccgg gagctgactt ttgaggctct ctggactggg actttgccat 16800 aaaactgctt atcagaaacc tcagataagt ggagtgccct gtgtcctggc cacgtactct 16860 ggggccagct cctccatcca ttcagtcctc ccacatccca tcttcaccaa gcagagtgga 16920 aagcggcagg tctctgcgag aaaaggaggc tctgtaccca gccttcagtg agataacttt 16980 agagtggggc cttgtattcc ctacacaggc cttgtccccg acacggcctc tttgcccttg 17040 cttctccttc tgctaagaat gctccttccc aactccttgg ctggacacac ggctcctgct 17100 ctgtgggcct gtcctgttcc cactcagagc ctcagcaccc cccatgtggc caagacacag 17160 cccctcttgc cttaggttgc cgtgggtctg tttccacatc tgtttcccct ccagactgca 17220 gggctcctga gggacagggc ctggggccaa gtctcctctg cgttcctgat gcctggtggt 17280 gtgccacgct ctccgtgggt gcacggtaaa tatctgccga gtggctagat gtatccatgg 17340 ttgctgaagg ccaagtgtgt tgaccagagg gcgtgggaaa ggctccactg gggtggctgg 17400 ggtagagata gaccttaagg ggtccagaag actccaccag ctggagggaa aggccctaca 17460 gagacaaggt actaggaaag gcccagagga cagaggaggg gccctgtggg gagcctgtgg 17520 gcaggaaggg cctgtgctgg gctttgaaac tggggactca ggtactgctc tagcgtgagg 17580 gtccttggca ttttttttgg tccaggaccc cctgaggtcc ctggggaatg agaacaggct 17640 cacacagcca ctttcaagag gtttgcaggt cccctgaggc ttagccacag acccctccca 17700 ccccagcaga gacacagcag aggcttctga gtggaggcca ggtggcttca gcctggggtt 17760 ctagaaggga gggaagagac acacccccgt gtgtcctcca gagcctcagc cttctccctg 17820 aatctcccct ttgcctcctc actgtaaccc acacctggct ctccttgaag gacccgcttg 17880 ccccgcagcc tcctgagagt tttctctccc agtccctctc ccactgggcc tggaggtggg 17940 gtagggtcct ccttgccccc agccttgaat cttctgtcac cctacaatac cacttgctgt 18000 caccccggtg ataggtatct acgagcctgg ggtgctcccc cacatcctca aagatgtgag 18060 cccctgactc agcgttgcct ctggttcctg accctccaca ttcacatgac cacgcgctgg 18120 cctttatcat cgccagtgac agcagctctc ccgtaaatac aagtttcaag caccccactt 18180 tccagtctct gccttctgtc tttccagttc attccctcta gtcctccacc cacacagtgc 18240 ttttccccat tgacagggaa ttgctcctgg ccgcttttcc cagccccatc ccgttcatgt 18300 gccttgcttt gtacccagca tggactccat ggcctgtcat tgtcactcct tcacctatac 18360 ttcctgcctc cttgcttctc tctctcttcg tcatcctggc atcaccaccc cagaactgct 18420 tggtttcagt tctccaccat ggctagggga acataccttt ttgctgactg ctctcacttt 18480 cttttttttt ttttttcttt tttttttttt tgacacagag tctcgctctg ttgcccaggc 18540 tggagtgcag tggcacactc tcagctcact gcaacctcca cctccggggt tcaagcagtt 18600 ctctctgcct cagcctcctg agtagctggt attataggca cccgccacca cacccggcta 18660 atttttgtat tttttttagt acagacaggg ttttgccatg gtggccaggc tggtcttgaa 18720 ctcctgacct caggtgatat gcctgccttg gcctcccaaa gtgctgggat tacaggcatg 18780 aaccaccctg cccggcccta ttgctctcac tttgaaatca cgacatgagc cacagtgagc 18840 ctatgatgtg gtttgacgtc tttctgcatt tctctgttca cccctctccc agaaggcatg 18900 cctccttccc tcccctcagc ctgcagcccc ttcccctcca cgcttactct cagctgatag 18960 ccctgctgct gtctcgctga gaaaatagaa atagccagag gagaattccc tctaccccat 19020 gcacacacct ctgtccccgc ctactccaca ttccctgttg tcagcaccga tgtgtttagc 19080 tgagggtggt ccccccattc aaggccatca tctcctgtca tctacttctt gacaccattt 19140 gaacaattat ctcccctctt tcctacataa cactttcccc ttttctacta tatatttttt 19200 gctatggatt gaaagtatcc ccccgaaatc atatgttgaa gccccaatcc ctcgtgtgat 19260 ggtattagga ggtagagcac tatgggggtg attaggtcat gaggatggag ccctcatgaa 19320 tgggattagt gcccttagac gaagaggcaa gaaagagatg atctgtctct gccatgtgag 19380 ggcacaacga gaagatagcc atctgcaaac caggaagtga accctcatca ggaactctat 19440 cagccagcac cttgatcata gacttcctag cctctagaac tgtgagaaat aaaggttgtt 19500 taagcctccc agtctgtagt tttctgttat aatgtcctga cctaagttat tttccatcgc 19560 tatacaatgg gttatttttt tctttaaaaa aaaattattt cttgactcta ctttttcctc 19620 cagagactgc ttatattcca ctgtcttcat tcttctcctc ctgttttctc tctttaaact 19680 tttcaaaaat agcaaaatat aacacatata gagaaaatga tatacagctc agtgatttat 19740 cacaaacacc tgtgtaacca ccactcgaat ctagagatag gacattacag caccccagaa 19800 gccctccttt tgtcccctct tcccaaagaa gggtaaccaa taacttgcct tttatgataa 19860 aggctttttt tttttgagat ggagtctcac tgtgttgccc aggttggagt gcggtggcgc 19920 aatctctgct tactgcaagc tctgcctcct gggttcacgc cattctcctg cctcagcctc 19980 ctgagtagct gggactacag gtgcccgcca ccacgcccgg ctaatttttt gtattttagt 20040 agagacgagg tttcaccgtg ttagccagga tggtcttgat ctcctgacct cgtgatccac 20100 ctgcctcagc ctcccaaagt gctgggatta caggcgtgag ccaccgcccc cggccaagat 20160 aaaggctttt ctatctggct tctttgttag cattttgttg gcgagattga tacgttttgt 20220 gtgcagctgt ctgtcatttt cattgcaata gaattccatt gtctactata tcacagttac 20280 tcattctact actgatgaaa atttgggtta tttctagttt ttttggtgca tacatgcata 20340 catttctggt agacatacct agaagtgaac tggctgcatt atgggccata acttgtcttc 20400 aacgttagta aataataccg cccgtctgtt ttccaagcag cagtaaatgg cactttctgt 20460 tacttcatat cttctgttac ttggtactgt caggttttaa aattttagtc attctgtcgg 20520 gtgtctagta gtatgttatt gtggttttat tttgcatttg cctgattgca aatgaagttg 20580 agcacttttt gatacagtga ttaaccattt ggatatcctc ttcagtgaag tatcagctca 20640 attctcttga ctatttaaaa aataaagttg tttatctttt tcttacagag ttttaggagg 20700 agttttttgg tacatgtgca gtccaaatac acaccttttg tcagtgatac gtgttcagct 20760 gcacacacgg tgttttgcct ttttattctt ttaatggtat ctattgataa acatgagttc 20820 ttagttttaa tgaagtcaaa tttgttcatc ttctttacgg tgtacttttt gtgtcttgtt 20880 taaggaagtc cttctctatc ttaaagcttt gagaagcatt ccttctttct cctattttct 20940 aaaaattgtt gaagatctcc ttccttcctt ccttccttcc ttccttcctt cctttcatct 21000 atctctctct cacccaggcc aaacttcagt ggcatgatca tggctcactg cagcctcaac 21060 ttcccaggct caagtgatcc tcctgcctca gcctcctgag tagccaggac tacaggcacg 21120 caccaccata cctggctaat tttaaaaatt ttttgtagag acacggtctc atcaagttac 21180 ccaggctggt cttgaactcc tgggctcaaa caatcctccc acctcagcct cccaaagtgc 21240 tgggattaca ggcatgagtc acatgtctag cctgtattct ttctttctta aatgtttgga 21300 agaattcatg ggtaaaacca tgtgaccctg gagtttattt gtgggaagct tttaaaacat 21360 ggattcgatt tctttaacaa atataggact atgagatttt catctcatgt cggtttgggt 21420 atgttgtgtt ttcaaggaat ttttctattt tatcgaagtt gtcaaatttg tctgcaggtc 21480 cccttatttc ctttttaatg tatggaggat ctgaagggat ggtactttgt gttgtctcta 21540 tttcttgttt ggttttgcta gtgatttgcc aatttaaaaa atctttccaa agaaccaact 21600 tttggctttg tttatttttt gaaaacctga gtagctggga ttacaggcgc ccaccaccac 21660 acccggctaa tttttttatt tttagtagag gtggggtttc accatattgg ccaggctggt 21720 ctcgaactcc tgacctcaga tgatccaccc gcctcggcct cccaaagtgc tgggattaca 21780 agtgtgagcc agtgtgcctg gcaagccgct ttttaaatgg caccaatccc attcatgagg 21840 gcagagccct gatgatcgaa ttacctccta atggccccat ttcttaatac tatcacactg 21900 agaattaagt tttaacataa gaattttagg aagacacaaa cattcagacc atggaacata 21960 tataaaacag ttgtcctacc aactaacaga atacacattc ttttcaagaa cacctatata 22020 aaaattatag acactgacct atgctggcca taaagcaaat atgaacaaat ttcagatacc 22080 tgaagagatt tagactatgt tctccaacta cagtgcaatt aagctagaaa tcaatagcaa 22140 aaatgtaact ataaatgagc ccgtatgttt gaattttaag caagatatat gtaaattatc 22200 tatggttcaa aaaagaagtc acagtgaaat ttagaaaata tcttgacgtg gcgggttgca 22260 gtggctcacg cctgtaatcc cagcactgtg ggaggccaag gcgggtggat cacctgaggt 22320 aaggagtttg agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa 22380 acattagcca ggcgtggtgg tgggcacctg tggtcacagc tacttgggag gctgaagcgg 22440 gagaatcact tgaacccggg aggcagaggt tgcagtgagc cgagattgca ccactgcact 22500 ccagcctggg caacatgagt gaaacttcat ctcaaaaaaa aaaaaaaaag aaaagaaaaa 22560 gaaaatatct tgacccaatg atgatgcaaa tactacgtat caaacttgta ggatgaactt 22620 ccacttctgg ccatgacaga ggagcctgta tcagactagc cttcctattt atatatttat 22680 ttttgagatg gggtgtcgct ctgttgccca ggctggagtg cggtggtgca atctcagctc 22740 actgcaacct ccacctcctg agttcaagta attctcctgt cccagccttc caagtagctg 22800 gaattacagg cgcccaccac cacacccagc tagtttttta tatttttagt agagacgggg 22860 tttcaccatg ttggccaggc tggtctcgac acctgacctc aggtgatcca cctgcccctg 22920 cctcccaaag tgccgggatt acaggtgtga gccactgcgc ctggccgtga gtgacctttt 22980 taatgtgttg ttgaatttga tttgctggta ttttgttgag aagttttgca tcaatatcca 23040 tcagggatac tggcctgtag ttttcttttt ttatatgtct ttgtctggtt ttggtgtcag 23100 ggcaatactg gcctcataga atgagtttgg aagttttctc tcctctgttt ttcataatag 23160 tttgagtagg gttggtatta gtttttgttt aaatgtttgg taaaattaag cagtgaagcc 23220 actgggtccc aggcttttct ttgctgggag acttttcatt acagctttga tctcattact 23280 tgttattggt ctatttaggt tttggatttc ttcacggttc catcttggta ggttgtgtat 23340 gcctaggaat ttatccattt cttctagggt tttcaattta ttggcatata attgttcaca 23400 gtaacctcta ataatccttt gagtttctgt gatattggtt gtaatatctc ttttgtcatt 23460 tctgattttt ttgtttgggt tttctctctt tttttgttag tctagttaaa gatttattga 23520 ttttgtttat cttttcaaaa aactttattt tgttgatctt ttgtattgtt ttgtttcaat 23580 ttcatttatt tctgctatga tctttattat ttcttttctt ctactaattt tgggtttggt 23640 ttgctctttt ctagttcttt aagagacatc attagattat tttgaagttt ttctacttac 23700 ttgatgtagg tgcttatagg tacaactttc ctcttagtac ttctttccct gtatctcaca 23760 ggttttgata tgttgtgttt ccattatcat ttgtttcagg atgtttttaa atttccttct 23820 taatttcttc attgaccatt caagagcata ttatttcatt tccatgtgtt cgtatggtgt 23880 gcaaaatacc ttgtttttga tttgtagttt tatttcattg tgatcacaga agatacttga 23940 tatgatttca aatttaaaaa gtaatttggc tgggagaagt ggctcatgct tgtaatccca 24000 gcgctttggg aggccgaggc aggcagatca cgaggtcagg agatcgagac catcctggct 24060 aacacagtga aaccccgtct ctactaaaaa tacaaaaaat tagccaggca tggtggcagg 24120 tgcctgtagt cccagctact caggaggctg aggcaggaga atggtgtgaa cccgggaggc 24180 agagctggta gtgagccgag atcgcgccgc tgcactccag cctgggtgac agagcaagac 24240 tctgtctcaa aaaaaaaata ataacaataa taataattta agacttgttt tgtggcctac 24300 catatggtct atccttgaga atgatccata tgctgaggag aagaatgtgt attctgcagc 24360 cgttggatga aatgttctgt aaatatctaa taggttcatc tgatctatac tgcagactaa 24420 ggctggtgtt tctttgttga ttttctgtct ggatgaactg tctaatgctg aaagtggagt 24480 attgaagtct ccagctatta ttgtattgag gtctatctct ctctttagct ctaagaatac 24540 ttcctttata tatctgggtg ttccagtgtt gagtgcatat gtatttaaaa ttgttacatt 24600 atcttgctga attgaccctt ttatataatg accttctttg tctcttttta tagtttttgt 24660 cttgaagtct gttttgcctc atagaatagc atagctattc ctgctcttat ttggtttcca 24720 tttgcatgga atatctttct ccatcccttt agtttcagtc tatgtgtgtc tttacaggta 24780 aagtgtgttt catgtaggca acagatcact gggtcttgtt tttctatccg ttctgccact 24840 ctgtgtcttt tgattggaga gtttagtcca tttatgttca atgttattac tgacaagtaa 24900 ggacttactc ctgccacttt attaattatt ttcttgttgc tttgtgatct tctttctttc 24960 cttcctgttc tccttttagt gaaggtgatt ttctctggtg gtatgtttta atttcttgct 25020 ttttattttt ttgtgtgtat ctgctgtatg ttttttgatt tgaggttaac atgaggcttg 25080 caaataatat cttataacct aatattttaa actgatgaca ccttaacact gattgcataa 25140 acagacaaac aagcaaagag aaaactaata aaaactctac attttaactt tgtcccccca 25200 ctttttaact tattgtactg tttatgtctg aaaagttgta gttattattt ttgattggtt 25260 cctcttttag tctttctact caagatatga gtggtctaca tatgacagtg ttataatatt 25320 ctatatgttt ctgtgtactt gctattacca ttaagttctg tactttcaag tgatttctta 25380 ttgctcatta atgtcctttt ctttcagatt gaaaaactcc ctttagcatt tagcagtatg 25440 tgaactgcat ttttcaagtc cagaatttct gcttgattct tttttttttt tttgagatgg 25500 agtcttgctc tgtcgcccag gctggagtgc agtggcgcga tttccgctca ctgcaagctc 25560 tgcctcccgg gttcacacca ttcttctgcc tcagcctccc gagtagctgg gattacaggc 25620 gcccgccacc acgcctggct aattttttgt atttttagta gagatgggat ttcaccatgt 25680 tagccaggat agtctcgatc tcctgacctt gtgatccgcc tgccttggcc ttccgaagtg 25740 ctgggattac aggcgtgagc cactgtgccc agcctgcttg attcttttaa attatttcaa 25800 tctctttgct aagtttatct gttaggattc tgaattcctt ctctgtgtta gcttgaattt 25860 cactgagttc cctcaaaaca gctattttga attctctgtc tgaaaggtca catatctctg 25920 tctctccaga attggccact ggtaccttaa ttagttcatt tggtgaggtc atgttttcct 25980 gtatggtctt gatgcttgtg atgttcgtca gtgtctgggc attgaagtta ggtatttatt 26040 gtagtctttg cagtctgggc ttgtttgtat ctgttcttct taggaagact ttccaggtaa 26100 taaaagggac ttgggtattg tgatctaagt ttttggtcac tgcagccata tctgcattag 26160 ggggcacccc aagcctacta atactatggc tcttgcagac tcgtaaaggt accacttggg 26220 tggtcttgga taagatctgg aagaattctc tggattacca ggcagaggct cttgttctct 26280 tcccttactt tctcccctaa aagtggagtc tctctctctg tgctgggccg cctggagcta 26340 ggagaggggt gacacaggca cctctgtggc cactatcact aggactatgc tggctcagac 26400 ctgaagccag cacagcactg ggtctcaccc aaggcctaca gtctccactg cctggctact 26460 gcctacgttt gcttaaggca ctagggctct acagtcagca ggtggtgaag ccagtgagat 26520 tatgtccttc ccttcaaggc agcaagttcc ccctaccccc atcctgggaa ggtccttaga 26580 tgccatctgg gaggcagggc ctgcagtcag aaaccttagt aatctcccca gtgctctatt 26640 ctattacagc ctggctgata cccaagccat aaggcaaagt ccttcctgct cttccctccc 26700 ctttccacaa gcagagaagt ctctctccat ggccaccact gccccagccc tgtggcagtg 26760 ctgccaggcc actgccaacg ttcattcagg gcctaagggc tcttgaagca actgaacact 26820 gccaggcctg ggactctccc tttagggtag tgggctcccc tctgacccag ggcaggtcca 26880 gaactgccac cccacagcca acacttagaa tctgggaccc caagagccct cttggtgctc 26940 tgccacactg tagctgagct ggtacctagg ctgatttttg ctttttatga aggtactttt 27000 ttgtgtgtat agttcaattt ggttttcctc cagggaggac aattggtgga ggtttctatt 27060 tgggcatctt tttttttttt ttttgagacg gagttttgct ctttcaccca ggctggagtg 27120 cagtggcgcg atctcggctc actgcaacct ctgcctcctg ggttcaagcg attctccagc 27180 ctcagcctcc ccagtagctg ggattacagg tgcctgccac catgcttggc tacttttttt 27240 ttcaattaaa aagtaaactt taatgtcgaa aatgcaaact tggggaaggc agaaagatca 27300 cacacaaggc tgtcgcttca cacttggaag gttgcacggc ggccaggaga ggcactcctc 27360 acttcccaga cagggcgggg gccgggcaga ggcgctcctc acttgccaga cggggcggcg 27420 gccacatgcc tggctaattt ttgtattttt agtagggacg gggattctcc gtgttggcca 27480 ggctggtctc aaactcctga cttcaggtga tccacgcacc tcggcctccc aaagtgctgg 27540 gattacaggc atgagccacc gcgcccagcc ctatttggcc atcttgcttt gcctccacct 27600 tcatttgata acagaacacc cccatgtaac caccaccaca atcaagatat aagacatttc 27660 tgttattctc caaatttcct tcaccctcct ttatagtcca tggcacccac cccatccctg 27720 ggcaacacct atgtgcttcc tttttttttt tccccgcagg aggacgtctt cctataccta 27780 tgtgcattcc atcactgctg cttttaatca tatagcgtgt agtcttatga gtctgacttt 27840 ttcacttagc ataatgcttt tgcaatttgt gattgagcgt tttcatatga ttctgttttt 27900 tctgtctctt agcttggaag ttatacactt tttagtagta actgttggga gatcgttctc 27960 caagagtctc tctcatattt ccatacatcc tgctagcaga gacattgact acctgttttt 28020 gtctgtttag tgttgctata aaggaatacc taagtctgag tattatacat agaaaagagg 28080 ttttatttgt ctcatggctc ctcaggccat acaagaaaca tggtgccagc atcttaccag 28140 tgtcacatgg caagaaagga agtgaggaag agaaaataag ttgttaactg gttgttaaaa 28200 agtgccaggc acgggccagg cacagtggct cgtgcctgta atcccagcac tttgggtggc 28260 caaggcgagt ggatcacctg aggtcgggag ttttgagacc agcctgacca acatggagaa 28320 accctgtctc tactaaaaaa aatacaaaaa aattagctgg gcatggtggt gcatgcctgt 28380 aatcctagct actcgggagg ctgaggcagg agaatttctt gaatccagga ggtggaggtt 28440 gcagtgagcc aagatcacct gggcgacaag agcgaaactc tgtctcaaag aaaaaaaaag 28500 tgccaggctc tttttaacaa ccagttctta caggaactaa tagagtgaaa actcactcac 28560 ccccacccca cagggagggc attcattcat gagggatcca cccctatgac ccaaacacct 28620 cccattagcc ccccacctcc aacattggaa tcaaaattta acatgaggtt tggggggcag 28680 aaatccaaat tatagcacta cctttgttcc aggctacctt caaaggatgt ttatacagca 28740 aacagccttg gaaaatagag ttagggtctc cttgtagagc acagggtaag tttgcttact 28800 tttcagcata atcaagattg tgtcttcttc tggagcaaaa ctttgacaac aacaagcggg 28860 gcaaagtctg aaaccttttg cacatcttga cagaacctta gagattacga gatttatctc 28920 tgccttactc aaggtccatt agtatcttcc agaacaataa gttggtctta gaatccttta 28980 gctatgtatt cccttccagg cttcgatgct gtgtgctgtt gttttattta gtttttattt 29040 taaacccaac aagacgctat accatcaata cttatttaga tccccccaca tatttaactt 29100 gccatggtgc ttcattcttt catgcctctt taagcatcca tctaggatcg tgttcattcc 29160 acatgaagag aaagcttttg gcccagcacg atggctcaca cctgtaatcc cagcactttg 29220 ggaagcctag gcgggaggat cgcttgagcc caggatttcc agaccagcct gggctacgtg 29280 gcaaaaccct gtctctactg aaaatacaaa aaattagcca ggtgtggtgg ttcacgtccc 29340 tgctactcgg gaggctgagg tgggaggatt acttaaacct gggaggttga ggctgcagtg 29400 agcgttgatt gcaccactgc actccagcgt gggtgacagg agtgagacca tgtctccaaa 29460 aaaaaaaaag aaggaaagaa aaagcttttg tatttccttt cgtgtaggtc tgctaatgac 29520 aaagtctctg ggttttttgt ttgactagag aaatgtttta attttaacct tattcttgaa 29580 ggttttgttt ttgttttcat tttgttttgt gtgctaagta cagaattctc attggcattt 29640 atttatttag agacagagtc ttgctctgtg gcccaggcta gagtacagtg gcacgatctc 29700 cgcctcccag gttcaagcga ttctcctccc tcaacctcct gagtagctgg ggctacaggc 29760 atgcaccact acacctgact aatttttgta tttttagtag agacggggtt tcaccgtgtt 29820 gcccaggctg atctcaaact cctgagctca agtgatttgc ctgccttggc ctcccaaagt 29880 gctgggatta taggcatgag ccgccgtgcc tggccagcat ttattttctt ttcagctctt 29940 taaggaagtc attccattat ctctgggttc cattgttgag aagccagctg tcattctaat 30000 ggttgccctt tgaagataac ttttttccct ctgctatttt aaatatttcc tccttgtatt 30060 ttattttcaa caggtgtaat gtggcacata tatcagcaat tgttatgtta catacaatac 30120 atattgtgca acatatgtat cattatcagt tattgctgtg taacaaacta ctccagaaca 30180 cagtggctta taaaaggaat catttattta gtccttaaat ctgcaatttg agcagagctc 30240 agtgggaaag cctcagcttt actttatatg gcaccatata aggtaactcg caactggcaa 30300 gttggtgctg gctgttggct ggtagttcat tcagggctca gggcccagga ccaagagtcg 30360 tcaccatggc ttgcttcagc ctccttgagg catgatggct gggtcccaag agaagagaaa 30420 tagaaacttc cagtctctta aggcattggt ctggaagctg ggacggcatc acttttgcca 30480 tattctgttg gtaacgcagt tacatcgcca gattaaaggg taaagagata caaattccca 30540 ctgctcagtg caaggaatat cagtgatttt agggggccct gttttaaaac ccctacaagg 30600 tgctaagcgt ggatttctgg ggctttcttg ttttttgttt ttttctatgt gttttttttt 30660 ttttttttgg tggtggggtc agggagggct tatagggtat gttaaatcag cagaataatt 30720 ttatttattt atttggagat ggagtttcac tcttttgcct aagctggagt gaagtagcgt 30780 gatcagagat ggagtttcac tcttttgcct aagctggagt gaagtagcgt gatctcggct 30840 cactgcaacc tccgccccgc tggttcaagc aattcccctg ccttagcctc ccaagtagct 30900 gggattatag gcacccaccg ccacgcccgg ctaattttta tatttttagt agacatgggg 30960 tttcgccatg gctagtctgg aattcctgac ctcaggtgat ccacccacct tggcctccca 31020 aagtggtggg attacaggta tgagccactg tgcctgacct ataatgtttt ttaaaaatca 31080 gttttggaaa gtttgcagac attaactttt caaatgtagc ttccatttga ttctttttct 31140 actctgcctc tgatcctcca cttatgtatg tatgctagat cttttattag atagtctgtg 31200 tctcttatgt actctactat attttgtatt cttctgtctc tccatgcttt attctgttta 31260 ctctttcctg attatatttt ttaattttgt aatttccatt tgatgctttt ttgatcattc 31320 aaaactcttc accaaaattc taaatttcct tttttaacct ctcgaatgtt atcacagtta 31380 ctttaaagtc tattttggat aacttaaata tttacagtcc cctcacagat atgtttcttt 31440 tgtatattgt ttctgttggt tttctttcat gtcttatctc cacatatgcc tggtgagttt 31500 tgattatgtg ttggtcattg tatttgcaag tatgctcgta gaaataatct ggaatggccg 31560 ggcgcagtgg ctcatgcctg taatcccagc actttgggag gctgaggcag gtggatcacg 31620 agatcaggag atcaagacca tcctggctaa catggtgaaa ccccatctct actaaaaata 31680 caaaaaatta gctgagcatg gtggcacgtt cctgtaatcc ctgctactcg ggaggctgag 31740 gcaggagaat cacttgaacc aggaggcaga ggttgcagtg agccgagatc acgccactgc 31800 actccagcct gggtgacaga gcgagactcc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa 31860 gaaataatct agagcctagt ttgatgtttc cttacaaaga atgaatttat ttttgcttct 31920 cccattgcaa ctaggataac ctcagtctag tttctggaac tgaaatcact tgaggctgaa 31980 ctgtggtctc tgtgagagcg tgtctgtgta tggctcaccc ttattcctag gctacgtagt 32040 tctttaggac cctaacccaa agtaagagat gttcatcagg gctctcctct tggcagacgc 32100 ctgggctcca gtccctgtgt cctgagcccc atgaggctgt tactggtgct gctcgtctgc 32160 ccatcctctc cagtaacagc cacctgaaca ctgcaattcc tgggtttcca tcattccttc 32220 cagcctggtg agtcatcagt gtcttttgag gtatccagtg cctgccagca gatgtttttt 32280 aataatttgt ccagcatttc tggctgtcct cagtaggtgg tctgtttcac attccctagt 32340 ctgctgttaa tggaaagtgg aagttctgag agagaatcat cgttcccgtc agcattcaaa 32400 tgcctgatta cactttcccc attcgctatc ctttccttta tttgctcctc accacagcaa 32460 aactcctcaa aaatatctat ctttgcaatc ttcagattct cacattgtgt ccttaaaccc 32520 actttaatca ggccttggtt ccctccattc cactaaaagt aatgttgtca aggtcgtgca 32580 tgcctccgtg atgctaattc cagtgatgag ttttcagttg tcattctctg acatatcggc 32640 aacacttgat gtggagtacc ctgatgttga gaggctggtg agatgaagga aaaacccagc 32700 caagggcaat gaggaatggc ttccttccgc ctcctccttc cctatctttg ttgtggtttt 32760 tgttgtttct gtaacactga ttacgttata gtttatttaa tgtacttcct tattctatgt 32820 ttttacttta gtcattttgt tgatctcctc cataataaca taggctcggg ccaagcacag 32880 tggctcacgc ctgtaatccc agcactttgg gagaccaaga tgggtggatc acttgaggtg 32940 aggagttcga gaccagcctg gccaacatgg cgaaaccctg tgcctactaa aaatacaaaa 33000 attagctggg cgtggtagca agcgcctgta atcccagcta ctagggaggc tgaagcagga 33060 gaattgcagg agaatcactt gaacccaggg ggcagaggtt gcagtgagct gagatcgcgc 33120 cactgcactc cagcctgggt aacagagtga gactctgtct caaaaaaaaa aaaaaaaaaa 33180 aggcttcagg agggcagagc attttatgca attgttcatt aattgacccc tccatttggc 33240 acagatcaca tgcccagtag ttaactgtta atcaacgcac tctctgtgga acaacaggct 33300 ggtcccgttg agccaacact ctctgggcac tgccttcact agactctggc tggctgctgc 33360 gcttgtagag gtatgcaaga caaaaccttt tggtatgtta aaagccctgg tcagaacttt 33420 aagtaaccat cactgacata taatggttat ggttttgaaa gtgcttttat agtggttttt 33480 catctgtcct cacagcacct ctgggaggta ggtaggacag gtacagttag tctcatgttt 33540 caaatggggc tcagagaggg aaaaggattt cccaagatca cacagcacct tagtagcaaa 33600 acttggactc aacctcaaca cttaaaaatg aacaaagctg gccgggcgca gtggctcatg 33660 cctgtaatcc tagcactttg ggaggcccag gcaggcaggt cacctgaggc caggagttta 33720 agatcagcct ggccaacatg acaaaactct gtctcttcta aaaatacaaa aaattagccg 33780 ggcatggtgg cgggtgcctg taatcccagc tactcaggag gctgaggcag gagaatcact 33840 tgaacctggg agatggaggc tgcagtgagc cgagatcgtg ccattgtact ccagcctggg 33900 caacaagaat gaaactccat ctcaaaaaaa aaaaaaagaa aagaaaagaa aaagaaaagt 33960 ttcccttttt ttaaatatct cattttaatg actgcataat agtttattac atagttatac 34020 cataatttat ataatatttt gttgctgtac atttaggtta aacaatactt acttagttcc 34080 tactatgaag aggggccaag tactcgaggc tggagctctc acatgtggca atgaaattag 34140 aaagtggata tggcagggct gtggtaaaat aaaatataaa aataataaat gatattacca 34200 aagaaaagga aagtttttca aaataaagga aaatctagac aagcatagtg gctcacacct 34260 gtaatcccag tacttttgga ggctgaggca ggaggaacac ttgaggccag gagttcaaga 34320 ccagcctggg caacatagca agaccccctg tcaacaaaaa taaaaaaaaa ttaggccagg 34380 cacagtggct cacggtggct agtgcctgaa atcctagcac tttgagaggc tgaggggggt 34440 ggattgcctg agctcaggag ttcgagacca gcctgggcaa catggccaaa ccccatctct 34500 actaaaaaca caaaaaatta gccaggtgtg gcagcacacg cctttaatcc cagctactct 34560 ggagactgag gtaggagaat tgcttgaacc cgggaggtga aggtcgcagt gaggtgagat 34620 tacaacactg cactccagcc tgggtgacaa aatgagactc tatctcaaaa aaaaaaaaaa 34680 ttagctgggc atggtggtgc atgcctgtaa tcccagctac tcaggaacta aggtaggagg 34740 atcccttcag cccaggaatt cgaagatgca gtgagtcatg atcacaccac cgcaccccag 34800 cctgggttac agagcaagac cttgtctcta aaaaacaaag aaagtctaac ctaaacgcca 34860 ttatgtcatt tacatgtgta taggggtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 34920 tgtgtgtgtg tgtgtttgct tgggagggga gtcgggctgg agctggccag gccgcagcct 34980 ggggaccttc tgattccagc agggatgtgg gccgagggtc ccgccttact catttcgggt 35040 cttgctcctc tgtcctggct ctacagactc ttccagctgg agttggaggc tgacgcccta 35100 gtgaatttcc atcagtattc ttcccagctg ctacccttct atgagagctc ccctcaggtc 35160 ctgcacactg aggtcctgca gcacctgacc gacctcatcc gtaaccaccc cagctggtca 35220 gtggcccacc tggctgtgga gctagggatc cgcgagtgct tccatcacag ccgtatcatc 35280 aggtgagcaa gggaacaaga ccatttggac aacgtgtgtg tacctgcttg tgtgggcagt 35340 gggggccgca ggcctcggtt cccctccata gtttgactta aaaaggagca gggcctggca 35400 tggtcgctca ctaacgcagt cccagcactt tgggaggctg aggagggagg atcacttgag 35460 actaggagtt caagaccagc ctggcaaaat agcaagacct tgtctctacg aaaaaattaa 35520 aaaattagca tgtgtctata ctcctagcta cttgggatgc tgaggcagga ggattgcttg 35580 agcccaggag ttagagttta cagtgagcta tgattgcacc actgcacttc agcctgggtg 35640 acagaatgag accttgtctc taggaaaaaa aaaaaaaaaa aaaaaaaggt gctgcgttgc 35700 taccagatta aaatgtaaga aggacaatgt aatgtttcca gagtccggag tgctcaccag 35760 gtgccaggtc tgtgggagct gatttctacc caccatgaaa ttgaattttc ccaaccccca 35820 caaggtatcg tcattttatt ttactttatt ttattttatt ttattttatt ttattttatt 35880 ttattttatg ttacgttatg ttattttatt ttgagacgga gtcttattct gtcacccagg 35940 ctggagtgca gtggcgcgat cttggctcac tgcaccctct gcctcctggg ttcaggtgat 36000 tctcctgcct cagcctccgg aatagctggg attacagtaa tgtgccatca tgcccgggta 36060 attttttttt tttttttttt tttttttgag acggagtctc gctctgtcgc ccaggctgga 36120 gtgcagtggc gcgatctcgg ctcactgcaa gctccgcctc ccgggttcac gccattctcc 36180 tgcctcagcc tcccgcgtag ctgggactac aggcgcccgc caccacgccc ggctaatttt 36240 tttgtgtttt ttagtagaga cggggtttca ccatgttggc caggttggtc tcgaactcct 36300 aacctcaggt gatctgccct ccttggcctc ccaaagtgcc gggattacag gcgtgagcca 36360 ccatgcccgg cccaggtatc atcattctag agagagctaa caggacttgt gcaggtcact 36420 cagccagaga gggtgaaaga ggggcttgga tccttggtgt ctgcccaggc tgctcttctg 36480 gaagctgaag gcctcctttt gtgaatccat tcagccagca gatggagcag ctgttgcccc 36540 tggtgagggc actgtcccca cccttgcaca gggcaccagt tggccatctt gtgcagaggt 36600 agccacacct cctctgagtc tctatcctag atagctcagt taagctccca ggcccagaac 36660 tcaaaacctc actgctggcc gggcgtggtg gctcacacct gtaatcccag cactttgtga 36720 ggccgaggcg ggtgtatcac ttgaggtcag gagtttgaga ccagcctggc caacgtggtg 36780 aaaccccgtc tctactaaaa atacaaaaaa ttagcaaggc gtggtggcgc acgcctgtaa 36840 tcccagctac tcgggaggct gaggcaggag aattgcttga acccagaggc ggaggttgca 36900 gtgagccgag attgtgtcac tgcattacag cctgggcaac aagagtgaaa ctccacctca 36960 aaaaaacaaa acaaaacaaa aaccctcact gcactttagt ttccaggcgt gcttttggac 37020 agcactgtct catctcacca tcactctgtc catttcctgg ggcaggcaca gcagcttgtg 37080 tcgataagga tattgacgcc cagaaaggtt aagtgaattg cctgaggcta gtgagatatc 37140 cgtgttctgt gaccctttcc tgtgccacca ccctgcccac ttcctgctgg tgtggccctg 37200 gggcccatct cttgtacgag tgagcaaact gaacagaaac ctgatacagg tcacatcaga 37260 tataattaca ttcaaatcag cccaactagc tgagtggcgg gaatgctctt gagctcaaat 37320 aaaccagaaa gtccgagttt ccgagtgcac ttccctgcat gtgcacttag gtccccctgc 37380 caccttgtac cacacctgct cacctctgct caccctggcc ctctcctgca gctgtgccaa 37440 ttgcgcggag aacgaggagg gctgcacacc cctgcacctg gcctgccgca agggtgatgg 37500 ggagatcctg gtggagctgg tgcagtactg ccacactcag atggatgtca ccgactacaa 37560 gggagagacc gtcttccatt atgctgtcca gggtgacaat tctcaggtgc tgcaggtgag 37620 cagggggagg ggcagggtga ctggtactga tacctcccgg tgtccacggt tccctttggg 37680 cccctaggct caggtgtcac tctcaggcct ctcactgtcc cccattggtc cttgaggcct 37740 gagctccttc atggagggtg cagacccctc cctggctctt ccctcccagc ccttttccag 37800 gagccacgtc ctcagtctct ttgcttgata aacctgggga cggcctccaa cccagcaaaa 37860 taaaccccaa aaaaggccca gctccagtca gttagttcag ttccactttt cctgagcaca 37920 aactgtgagc cagatgcctg cctggagggg ctcgtgggcc agcgggacct gggcttaacc 37980 ttttgcaagc ttgtccaatc tgtggccctg caggctgcat gcagcccagg acggctttga 38040 atgtggccca acacaaattc acaaactttc ttaaaacatt atgagatcca tgcatggacc 38100 ttttttttag atcagctgtt gttagcgttc atgtatttta tgtgtgaccc aagacaattc 38160 ttctgatgtg gcccagggag gccaaaatat tggacaaccc tattttacag gaattcaaac 38220 caagcaaacc aaactgtctc cagttcctgt aggcatttgt gccagaggcc ctcctgttaa 38280 cgtttttgat tacctgggca aaagatgtga ttttttaatt gtgggcaagc agatggggtg 38340 atactattaa atagctattt catatattag attgaacaga aaaggcagga tgttggatta 38400 tgttttattg tctagaatta gaaaattctt ctaaagatca tgttctctct gccttggcat 38460 cagaatgcaa ctacgttata ccagaaaatg tggattcctt tttttttttt tttttttttt 38520 tttttgagac agagtttccc tatgtctccc aggctagagt gcagtggtgc gatctcggct 38580 cactgcaacc tccacctcct gggttgaagt gatgctcgtg cctcagcttc ctgagtagct 38640 gggattacag gcatgtgcca ccatgcctgg gtagtttccg tatttttagt agggatgggg 38700 ttttgccatg ttgcccaggc tggtctccaa ctcctggcct taagtgatcc acctgcctca 38760 gcctcccaaa atgccgggat tacaggtgtg agccactgtg cccggcccag aaaatgtgga 38820 ttcatatctt atgcttcttg ctctcaggga agggaattct ccacccactg ttgaactcca 38880 ctttcaccga gaatcctctg cgcacccagc atggtgccgc gcagaccctg cctccatgcg 38940 ggggtgtgca cacccgcttc tcgatagtca tgcacgactg cagctgtcct ttatccggtg 39000 ctgattggtg cctgaccttg aactgagtgt tgtatgtgtt ccagttaatt cttagaatac 39060 actcatgggg tggaggtcat tattcccctt tcctctccga agaaactgag gttcagtgtt 39120 gttaattaag aagcgtgccc agggagggcc acaaagctga gagagtgggg cttgacctta 39180 cattggcctc gatgccagcc ctttgctctt cctgctgtgt ctctggcctg ccttcaggtc 39240 cagtgggact gctgtgaggt gaccctggtg tcacatgggt acactgtggt tgtaatgaac 39300 ctcatgggga cagcactgag ccaaaagtga gacagtaaaa ccctcctagc actttctttt 39360 ttttgagaca gcatctagct ctgtcgccca ggctggagtg cagtggtgtg attgtggctc 39420 actgcagcct caacctcctg gactcaggtg atcctcccac ctcagcctcc tgagcagctg 39480 ggactacagg ctcgtgccac cacatgggct aatttttttt ttttttgaga cggagtcttg 39540 ctctgtcgcc caggctggag tgcagtggcg cgatcttggc tcactgcaag ctctgcctcc 39600 cgggttcatg ccatcctcct gcctcagcct ccagagtagc tgggactaca ggcgcccgcc 39660 accacacccg gctaattttt tttgtatttt tagtagagac agggtttcac cgtgttcgcc 39720 aggatggtct tggtctcctg accttgtgat ccgcctgcct cggcctccca aagtgctggg 39780 attacaggcg tgagccacca cacccaggcc acgccagcta atttttaaaa tttttttggt 39840 aaagatgggg tctccctatg ttgccgaggc tggtatcaca ctcttggtct caagtgatcc 39900 tctcacctcc cagtgtgttg gaattacaag catgagccac tgcgcctggc ccctggctct 39960 ctttttaata ctgctgttct ttcattgaaa acatagtacc tgcatgtaga aaaaaatcag 40020 tcaggacaga atgaaaagca agaatcattc ccacctggac ccccagtgcc catcctgaga 40080 tacagctacc ttttaagttc cttgactcag aaatgtttca tgtgagccac agtcatgcca 40140 ggtcgacccc ttccctccca tccacactgg cacttggctt tcctggtgct tctctttacg 40200 ttcttttttt ttagacagag tttttgctct attgcccagg ctagagtgca atgatgcaat 40260 ctcagctcac tgcaacctct gcctcctggg ttcaagcgat tctctttcct caggttcctg 40320 agtagctggg attacaggtg cctgccacca tgcccggcta atttttgtat cgttagaaga 40380 gacgacattt caccatgttg gccaggctgg tctcaaactc ttgacctcag gtgatccacc 40440 caccttggcc tcccaaagtg ctaggattac aggcatgagc cactgcgccc ggcctcttta 40500 cgttcttatc agcatcctat ggagtcaaac ttcccttcct ctttcatcag ctccttggaa 40560 ggaacgcagt ggctggcctg aaccaggtga ataaccaagg gctgaccccg ctgcacctgg 40620 cctgccagct ggggaagcag gagatggtcc gcgtgctgct gctgtgcaat gctcggtgca 40680 acatcatggg ccccaacggc taccccatcc actcggccat gaagttctct cagaaggggt 40740 aagacctcct cccctcccca tctgctggcc tgctgggctc cagctctcct gtcacctgtc 40800 ccgtgcctga ggcaagcagt atccaccata gatcttagca tctccagcct cagagcagag 40860 gccccgggtt cacaagcctc attaggcccc gcctggccca aagaaggtgc tccatcattg 40920 tatcatgggt aggaatctcg gttcacaagt tcagctgtgc aaggaggaga ctttaaaatt 40980 tttctggctg ggcgcggtgt ctcacgcctg taatcccaac actttgggag gctgaggtgg 41040 gcagatcacc tgaggtcggg agttcgagac cagcctgacc aacatgaaga aaccccatct 41100 ctactaaaaa atacaaaatt agctgggcgt ggtgacacat gcctgtaatc ccagctactt 41160 ggaaggctga ggcaggagaa tcggttgaac ccgggaggcg gaggttgcag tgagccgaga 41220 tcgcaccgtt gcactccagc ctgggcaata agagcgaaac tgtctcaaaa aataaataaa 41280 taaataaata aataaataaa taaaaataaa tttatttaat gtaccttaat tcctaggagt 41340 gaatctccct ctataaaact ggagacaacc aatcttagcc ctctttgggg tctcctcttt 41400 ccagctcccc tccaggtggt gctccagttc tggagtctga tgcaacccca aggcccacct 41460 ggcccctcct ctaacccagc ccagcctgat ctgaacagcc ccccgatggc ttcagtgctc 41520 cttggtttct tgtgtgccca gctcaaagcc tgggaaatgc ttggtaaggt ggccgagtgc 41580 ccgtccacct agatgcctct ttcctttctg gctggtggca cgttgggagg caggtacagg 41640 ctcatgtttt gggagcctgg gaacccctgg tgtcaggaaa attgtcttca aaggggtagg 41700 ggatgagata aaaagataaa tgaatatctc aaatctcaaa atactatcta ggccaggcgc 41760 ggtggctcac acctgtaatc ccagcagttt gggaggccaa ggtgggtgga tcacctgaag 41820 tcaggagttc gagaccagcc tgaccaatat ggtgaaaccc tgtctctact aaaaatacaa 41880 aaattagcca ggtgtggtgg tgtgtgcctg tagtcccagc tactcaggag gctgagacag 41940 gagaattgtt tgaaactggg aggcagaggt tgcagtgatt tgagattgtg ccacagcact 42000 ccagccaggg cgacagaaca agactccatc tcaagaaaaa aaatactatc taacaggaac 42060 aacatctaga atgaatgaat gagccaatga gtccttcgat cctggcactg tgctaaagca 42120 ccagcctcac cgaaatgaat aagccactcc aggacaggag acaccccagc cacccagaat 42180 ctcatagcgc agtgccagac aagcagtgag aaggcatgga cgggcagcac tcaggcaggg 42240 aggctggcac ttgtgcctgt cctcaggttg ttccttcctc tgggagctgc atccaccttg 42300 ctagccccat ggggcattgc tagcccttct aagggctgga ccaagcctgg acaaaccctc 42360 ctttccttga atctcccaag cacagccaga atgagctaga gctttcagaa atacttgacc 42420 aaacagagaa gataccaaca cccccagccc cgcagtcagg ttccccattt gcaggttgtg 42480 acctgagtgg acaccaggtg atgctcttct ctacccagag ggctggcaca tggccccact 42540 cttgcttgtc tccagccacc ctgagctgta ctgggcagga gctgtaattg accagttttc 42600 tctctcttca aacaacctca gccctcaccg gtccctctgt ccccacacag gccatcctgg 42660 gtgggtccag cgtcccatca cataagagga tctttgcaca gaagacaaag gaagaggctc 42720 ctaccttgag tggagcaggg agatccctta tcgacttggc agccagcaca gccctactcc 42780 aagaatccca cggcctcagt gttactgaca agtggttccc tcacagctca gactaggagc 42840 tccctcgctg tttttctttt tcttttcttt tctttgtttt gtttttgaga cagagtttca 42900 ctctgtcacc caggctggag tgcaatggca tgatcttggc tcactgcaac ctctgcctcc 42960 caggttcaac tgattctcct gcctcagcct cccaaggagc tgggattaca ggtgtgcgcc 43020 actatgccca gctaattttt gtatttttag tagagatggg gtttcatcat gttgaccagg 43080 ctagtcttga actcctgacc tcaggtgatc tacttgcctc gccctcccaa catgctggga 43140 ttacaggcgt gagccaccgc gcctggccac ctctgctgtt tttcaatgca gctgaccttt 43200 cccacaaagc tcggtttgtg cctgcggttc tcagatggtt aagcccctgc tgaggaaggg 43260 aaacagataa tcccagacag tgagatggag gctgagactg aggatgcctg ggggctgcac 43320 taggagctca gagacggcat caggcagctg gagttcaggg aaggtttggg ggcagcagag 43380 cccgagccaa gccatggaca ctagccggag ctggccactc gggtgaagac ttggggggct 43440 ccagagggcc cagggaaaga agctgggcct gactccatgt gcctagtgct gagcaccaag 43500 cgctggcata agggctgtag ggagctgggc agcataaatc ctgggcctgt gctgggcacc 43560 cctaataata gagaacagca gctgaacact aacgctgtcc cagccaccgg cttagccttc 43620 atgtgggcta tttcactata gcccagagag ctgatgtatt ttattccccc tctaaagctg 43680 agggccaagt gtggtggctc atgcctctaa tcccagcact ttgggaggct gaggcaggag 43740 gatcgcttga gcctaggagt ttgaggctac agtgagctat gatcatgcca ttgccctcca 43800 gcctgggtga caaagggaaa tcctgtctca aacaaacaaa caaaaaacag atgaggaaac 43860 cagggcttag agagattaag taatttgctc aaagtcaggc agtcagtaag tgccagacct 43920 gggaattaaa cccatgtggg ctgtcctagt aattgcacta aaaaaaaaaa aaaaaaaaag 43980 ctttattgag atatagttcc tattccatac aatccaccta ttgaaagtat acaatgattc 44040 tgatatatta tgtttatttt ttccaactgt gatagattat atataacata aaatttgcca 44100 ttttaaccac ttttgttttt tgtttttgag acagagtttc gctctcgttg ctcaggctgg 44160 agtgcaatgg tgtgatctcg gcttaccgtt acctccacct cccgggttca agtgattctc 44220 ctgcctcagc ctcccgagta gtagctggga ttacaggcat gcaccaccat gcccagttaa 44280 ttttgtattt tttagtagag acagggtttc tccatgttgg tcaggctggt ctcgaactcc 44340 cgacctcagg tgatccaccc accttggcct cccaaagtgc tgggattaca ggcgtgagcc 44400 accacacccg gcccatttta accactttta agtgtacaat tcggtggttt taatcacatt 44460 tacaatgttg tccaaccatt acccctttct aaaacttttc ttatcacctc aaacagaaac 44520 tgtaaccatt aataactccc gattctcccc tccccacagc ccccgataag tcccatatcc 44580 tctctgtctc tcagaacttg cctgttctag aacctcgtat cagtgaaatc atacagcgtt 44640 tgtccttttg tgcctggctt atttcactta gcatgatgtc ttcaaggctc atccatgttg 44700 cagcacgtat cagaatttcc tttcttttta aggcagaata ctattttgtt tatccattca 44760 gtccttcact gatagacgcc tgggttgttt ccaccttttg tctgttgtga agaatgctgc 44820 tgtgtacatg ggtctgcaaa tgtccattca agtccctgca ttccgtcctt tgatacttcc 44880 gacctcaggt gatccgcctg cctcagcctc ccaaagtgtt gggattacag gcgtgagaca 44940 ccgcgcccag ccagaaaaat tttaaagtct tctccttgca cagctgaact tgtgaaccga 45000 gattcctacc catgatacaa tgatggagca ctttctttgg gccaggcggg gcctaatgag 45060 gcttgtgaac ccggggcctc tgctctgagg ctggagatgc taagatctct ggtggacact 45120 gcttgcctca ggcaccgagt agaaagcaat tgtgctcttt cccactctgt catactgctt 45180 cctatagcat gcaggtcact gtgtgactca ctgacagtgt ggtaaggagg tggagggtgg 45240 ctgtgtgcca ggcacagaac tgggctctga aatgcctggt cttccttaca ccccagccac 45300 tcctctcagc actctctcca tgttgctcag cttctggagg ccaccagtga ggaagctggg 45360 gcttgacttc aagcccatct gactctaaag ccagtaccag ggagggtgat cattgctggt 45420 ggcagcatca ggaaggcttc ctggaggagg gggttagttg tgccttgagg ggctcagtac 45480 agccttaacc atggtgagtg tgctggttgg gtttgctgaa ctttgtttgc agtgatgctc 45540 tctgtaagca gtagatgaag ctccaagtcc tggcttcatc ccacgccacg cagaccctag 45600 cttctgctca ccaaccgggg cccctctctc accaggtgtg cggagatgat catcagcatg 45660 gacagcagcc agatccacag caaagacccc cgttacggag ccagccccct ccactgggcc 45720 aagaacgcag aggtgagtgg atcctgaggt gggtgggtgg ggcagggggc cgggccccgc 45780 aggtcctcag ggccctcagg aagcaggttc ccgagaggac cctgaggcta gcacttgggg 45840 gctgaccttg cagtgggtgt gcaggcagag aagtccatga agagctggga tcctggcagg 45900 gctctgctgc tctctggctg ggcgggggga tccgccctgt gcctcagttt cctcatccat 45960 gaattctgct ttggcgatgt gatgaagtca gcaaggcagt gtgtgacaaa actcggcaca 46020 aattgtaaag tgcttcccaa atagaaggtg gcgtgatgat gattgtatta ttagtcagga 46080 ttctctagag ggccagaact aacaggagat atatatatag agagagaggg gggtttattt 46140 ttttatttta ttttttttga gacggagtct cgctctgtcg cccaggctgg agtgcagtgg 46200 cacaatcttg gctcactgca agctctgcct cccaggttca tgccattctc ctgcctcagc 46260 ctcctgagta gctgggacta caggcgcccg ccaccacacc cggctaattt ttttgtattt 46320 ttagtagaga cgaggtttca ccgtgttagc caggatggtc tcgatctcct gacctcgtga 46380 tctgcctgcc ttggcctccc aaagtgctgg gattataggc gtgagccacc gtgcctggcc 46440 taaaggggga tttattaagg agcattaact cacacgatca caaggtccca caataggcca 46500 tcgcaagctg aggagcaagg aagccagtcc aagtcccaga gctgaaggac ttggagtctg 46560 atgtttgagg gcaggaagca tccagcacgg gagaaagatg taggctggga ggctaagcca 46620 gtctagcctt ttcacatttt tttctgcctg ctttatattc tggccgtgct ggcagctgat 46680 tagatggtgc ccacccagat taagggtagg tctgccttcc cctactgact caaatgttaa 46740 tctccgttag cagcaccctc tcagacacac ccagggtcaa tactttacat ccttcaatcc 46800 aatcaagttg acactcatta ttaaccatca agatgatgat gatgatgatg gatgatgatg 46860 gtggtggtgg tggtgctggg cctagctgca cacacaggga gcctggctgg gcatctcctc 46920 tgagttcttc ccctaagcta agctctgtcc tcctgggagg atggaggcct ctggtctcat 46980 caccaggaag cacagcttga cagagaatgg gatgccccag agttcatggc ctacttctca 47040 atgtgatggg ccaggaggga gggcactgcc agcctcaggg tacctctgca ttccaggagc 47100 cggacaccga gtcaggccct tccgtgtagg tttggtcact cattgtcatc acatgctgtg 47160 aggtggggac ttgcattccc atcttcacag aagccctgaa gtgcacatgg ctacagtgac 47220 ccatccaagc gcaggcagct gggatgagtg aagctgggct ttgggctcag gtctgtgact 47280 ccaaagccag gctcccctcc ctgttcccaa ggctctgcca gggatgtgca ttgccaaggc 47340 ccagtgagag gtgatacctt tgcagggttc cagggaggga tgcagggagc ggggtgcttc 47400 aggacaggcc ccagaggagc ctttgcaagg agagtgggtt ctgttggcac taggaaagga 47460 ccagaagcct cttaaaacag tcagcacatc tttggctttt tgtccctagt cttgggatgc 47520 tgatgggatg ggaggcagtc taggcccccc tagaactggg gcctgcccct ggaatcctct 47580 tgctagcgct ttccaacatc ccagtacctg taggcctctc agagcagaag tggcagtgcc 47640 cacgtgtccc ggggtgcggc agctccccga gtgcctgtgc taactcagcc tgaccgtctc 47700 ccgcagatgg cccgcatgct gctgaaacgg ggctgcaacg tgaacagcac cagctccgcg 47760 gggaacacgg ccctgcacgt ggcggtgatg cgcaaccgct tcgactgtgc catagtgctg 47820 ctgacccacg gggccaacgc ggatgcccgc ggagagcacg gcaacacccc gctgcacctg 47880 gccatgtcgg tgagcccagg accgcgtgtc ctgccctgtg gggccccggt gggaatgcag 47940 gagggctgtc cggactggag ccctcctccc cactgtctcc ctcctatcgt cagctgctcc 48000 aaatcatctt cccacccaaa gctcagttca gaccctgact cctccctcag gaagccttcc 48060 aagtccccct ctcagcccaa aataacacca cctcctgtgt gctgtggtac cttttccacc 48120 tgtgccccca tttggcactt gcatggcacg agagggaaaa atgacagctc tggccataga 48180 ctggccctga gcaagccatg tcacctgtca gcctcgattt cctcatctgg aaaatggtgg 48240 tgtcaatact tatctggaag agttgtggaa aagttatggt ggacacttgg tcaccctcta 48300 cagaatgtct aagtgacaca cgcagtacct ggcccgagtg agcatcctgc ttcccttcac 48360 tggtccgcgt gttgctctca tcttccctgg gaggctgtaa gatcctggca ggcaaagcct 48420 gagtgtgcag cccagcagcc tgtggcaggt gccaggcata ttcgttggtt tattatagaa 48480 cacagttcct gccctcaaag cccttactgt ctggttacac atctagagta tgaacatatt 48540 aaacctagca gaggaggacg ttactgtgtt acagacatat gtgtgtgcat gtattctttt 48600 ttttcgagat ggagtctcgc actgtcgccc aggctggagt gcagtggcgc gatctcggct 48660 caccacaacc tccgcctccc aggttcaagc gattctcctg cctcagcctc ccaagtagct 48720 gggactgcag gcacgtgcca ccatgcccag ctaatttttg tatttttagt agagacgggg 48780 tttcaccacg ttggccaggc tggtctcgaa ctcctgtgtt ggccaggctg gtctcaaact 48840 cctgacctca tgatccaccc gccttggcac cccaaagtgc tgggattaca ggcgtgagcc 48900 accgcgcccc accgtgtgca cgttttctat agcaataccg tatgtacaga agtctgttta 48960 ctgagtctca gtgacagaac atgagagacc cccgggactc taaaagctga ttcaggacac 49020 attcagagac atcctgtttt ataccaaagg tcaaaacttg gaagagatgt tgttgcccaa 49080 aaggctgaaa ctataaatag tttcattttt gaaactgtgg acatttttag cactgggtgg 49140 gcccttggaa tgatctaact cagcagttct caagtttttt ggcctatttg atccctttat 49200 actctgaaaa attattgagg atcacaaaga ttttttaatc atctggattt tctttatcta 49260 tatttaccat tttagaaatt taaagtgtga aaatttcaaa atattaattc atttaaaaat 49320 aacaataata aatatatatg cctactatgt acccacaaaa attaaaatta aaaatttttt 49380 aaaaaactta aaaaataact ggccagctgc agtggctcgt gtacaccagc actttgggag 49440 gccaagatgg tcagatcacc tgaggccagg agttcgagac cagcctggcc aacatggcga 49500 aagcccatct ctactaaaaa tgcaaaatta gctgggtgtg agacctctga aagggtatca 49560 cgtgttcaca ggggtcccca ggtcttactt taagaactgc tgatagactt tggtcatctt 49620 catcccatct tcacagtttt tggcatattc atcaaccacc tttacaatta tgtacttaat 49680 cacctcttca aattggctta cttttattta tttattttga gacagagttt cgcttttgtt 49740 gcccaggctg gagtgcaatg gcacgatctt ggctcaccgc aacctctgcc tcctgggttc 49800 aagcgattct cctgcctcag cttcccgagt agctgggatt acgttcatgc gccaccacac 49860 ctggctaatt ttgtattttt agtagagacg gggtttctcc atgttggtca ggctggtctt 49920 gaactcctga cctcaggtgt tccacctgcc tcagcctccc aaactgctgg gattacaagc 49980 gtgagccacc gtgcccggcc tggcttactt ttaaactgac ttaaatttat tttgagagaa 50040 atgttgcatc actaccataa atggccatcc atccatccag tgccaaccat aaaaggtaac 50100 caatgcaggg cgcggtggct cacgcctgta atcccagcat tttgggaggc cgaggtgggc 50160 gaatcacgag gtcaggagtt caagaccagc ctggccaaca tggtgaaacc ctgtctctac 50220 taaaaataca aaaaattagc tgggcgtagt ggtgggcacc tgtaatacca gctactctgg 50280 aggctgaggc aggagaattg cttgaactcg gtaggtagag gttgcagtga gctgagatca 50340 cgccactgca ctccagcctg ggtgacagag tgagactccg tctcaaaaaa aacaaaacaa 50400 aacagtaaca acaacaaaaa atcacagtta atgctgaaaa cacacacata tctgtagcca 50460 tagaagtcct aatagaatgt gaagaactgt ttggaaaacc ccattctaga cccacagctt 50520 cattctctag gcagggaaat cgggtcccaa gaagaggaag gtgacaaggc cagagcccac 50580 agttccaggt ggccagtgtg tcctctgaaa ccatgccatg ctctgcgccc ctgcccggcc 50640 tccaggtgtg gaaatgtgtg acgttcttcg tggggacact cccccgaagg ctgccattag 50700 gggcacgtgt gttcggttac ccctcaggga tgacaccctg gactgggtgg gcaagatggc 50760 cttccctgcc ctcacccacc ctctccttgc tactcctgaa agccatcagc agctgaccta 50820 ctgtgtcccc atggctcctg ggtcggggtc acagatgcac tgcatctttg gaggcggagc 50880 tggcagagca ggggcgagtc acttctagtt tctgcctggg ttcttacacc ctcccctgtg 50940 ggtgctttta agcctgtttg tcaggtcccc agccatcaaa tggcagactt tggactacca 51000 gattgacttg ggtgggggtg aggagggttc cagtgtctcc ccgtgctgag gagaggcccg 51060 ctgggtgagt tgacaggttg gggctggcag ccaggggccc ctttgttctt cacttccccg 51120 gcagttgcac gtcttatcct ctctccatct ccaacagaaa gacaacgtgg agatgatcaa 51180 ggccctcatc gtgttcggag cagaagtgga caccccgaat gactttgggg agactcctac 51240 attcctagcc tccaaaatcg gcagacgtat gtgctctgca ctctggggct ggagtggggt 51300 gagggttggg tggatacagg gaccgaggag gagggaagtc ccaggcatca ggtgccagca 51360 tcctgtgctg acccgccctg ctcagagctg ccaggagcac ctcgtactca gagagggggc 51420 ttccttaccc aagcacaagg gccctgagca gtagatgcag gcccaccacg gggcttgcta 51480 taccagcatc ctctcccgtg cagcgccccc cacctcaggg agctaagatt cccagcaggg 51540 gtggggacag gagagccagc agcagccttc tttccccacc gttggggaat tcactgatgg 51600 aaccactggc tagaatgacc aagggtgcat gcactacact gcacgagctc tgggcatgcc 51660 tgtgtgggca gagtgcatgg ccatgtgctg cacgctcgca cagtgagcct tagcttcaga 51720 gctggacaga gcagatcctc ctcaccagag gcctggagcc cgctctggag ggtttcagca 51780 gctgtggggg tcactgtggg caggtacctg atggcagcag tgtgtgtgtg ccagtgcaca 51840 cgtgtgtgtc tgcatgcacc gcatctatga cctccttttc agcaatccca tccatcaaat 51900 ggagtaggtg ccccctggct ggctcattag tccctttgga accctgaact atcacttagc 51960 atttttgaga atcactgaat gtggccatgg gattcctgct ttcttggctg gagtagaagg 52020 aaagaagggg ttcccacagg catcgggcag ggtgacactg acctcctcct cccatatcct 52080 ccttccctgg ccttaggaag ctgcttggga tgtaccagcc tgggggcagc actccctgtc 52140 agggtgggct gaggcctctg gagcagccca cgtgcccagg gcctgggagc tgggacaggg 52200 gacggagtgt ggaaaggagg ggcctctcct ttgcatgttg acatcatttc tgatcctgtg 52260 ccatctcccc ttccccaaac catgacaagt tgtcaccagg aaggcgatct tgactctgct 52320 gagaaccgtg ggggccgaat actgcttccc acccatccac ggggtccccg cggagcaggg 52380 ctctgcagcg ccacatcatc ccttctccct ggaaagagct cagcccccac cgatcagcct 52440 aaacaaccta ggtaggcctc gcctcccgac tccctcttct ccagctggtt cccaggcccc 52500 tggatcacag caggccccac caaagcaaca ggatccctag gacggaagtc actaaaggaa 52560 gccggggagg atggggccgg agttgtcagc ctctaggcag ccagcaaccc acagatccca 52620 gaggcctagg gtgtccctgg ctgatgtccc atgctccgtt gcccctgaga ggatgtgtcc 52680 tgcacacagt ctcaggcctg ggcatttcca gtggcaccca tgaggtggct gctgttactg 52740 ccctcctgtc cccagggcgt tcagggtggg cagtgaggag tgctgtcggg ggactggggg 52800 cccgggccct ctggcttcac ctgtccctct ggcttcacct ctatcacact gtttgaatct 52860 cggttgggcc tgagacctgt gtgccccctt cctggccccg tggtgacttt cagacgttga 52920 atgtggccag gcagccagca tctgccccgg tgcattcctt ggtgcccagg gagacatccc 52980 cagactagac aggacccttc tctggacttt ctgtgggctg gcccactcag ggacctccat 53040 tgtttgggag cggggtcccc tgaacagagg gcagccagga gccagtcctt ggtgagccag 53100 acttcactgt ggaggcggca gcaccttcag ccagggtagc ccgaccaagc ctggaactct 53160 ggagaggcac cgtgactgtg gcttctagtc ctggctccac ctctctctcc cctcccagga 53220 actccccctc cacctcccat ctttatctct caattttgca ggcagtcacc caagccaggc 53280 cggatggtgg gcctggggtg cggcgtcaga tgggtaacgc cctgggcctg gagaggccac 53340 cgagcctagc catgcggcat tagctctagc tctcactccc taatccgtcc ttcttagctg 53400 cgcacacacc acacgccccc tcccctgcac cctgtccccg gcctctctca gccactcttc 53460 tgcttccctt gttcactgtg gagccgtgtg ccctggggag ggggagacac cgcttcgcag 53520 ccctcggttc tgctttgctg cttctagact ctgcacagtg gtggggggct gtcagagttg 53580 gggtcacgcg ggctgctgca ccaggcacct ggggactggg ctgcttgtca ggaggggcag 53640 ctagtcagtt gggtggacgt caggcaggcc ttggacacaa aggaagacat ggacagagtg 53700 gatggtgggc ctgatcccgg aggccactgg gatttccaga cctgggatca ggacgaggga 53760 tgtctccttt catccatgga cttaaacccc gaggaacgtc ctgactcagc cttttgacta 53820 aatgaccttg ggtgaattat ggaccctctt agagcctcac ctgtcaatag ggaataagaa 53880 ttcttaggcc ccaggtggtt attgcagcat cggctccgat gcaagaagaa gcactttgtc 53940 tgaagaggac acgcaagggt attcatgcct tggggtttca agaggaagag attgagggga 54000 acctgggagc tggctgggca gggtggggag cccttcccag agcagtgggc ccccctttcc 54060 actccagccc atttctctcc tgtggcctgt ggctcagctt tctcctggga cagagtcctt 54120 cctgtgggga agggacagat gacaggggga gtggggggat gagggcgtgg ccgtgggcga 54180 ggcacagccc aggtttgatc tagggacctc tggggtagca gggcttgggg acccacctga 54240 ccacagcatg ccctgctctg tgcctcacag aactacagga tctcatgcac atctcacggg 54300 cccggaagcc agcgttcatc ctgggctcca tgagggacga gaagcggacg taagtggatc 54360 gagatcgggg gcagagcggg gaacgtgtgc gctttcctgc ccctcaccca ccccctgttg 54420 tgggctcagg gctttactct ctgcaccctc acagccggcg aagtggctct gctcctagtc 54480 ttatagttct cagcagagac agaacatgga gagcctgtgt cgctcgttca ttaatgctgg 54540 gcggtgcagg ctctggagac cagcccaggg tgccaggctt cttggacttg gtggggctgg 54600 cgcagtgttc tggcctccct ccctgatctc cttatggcta gaagaggggg agatgcctgg 54660 gtcccccctg gggagatctc agcagctgct tcccactgct cctactggct tccttaacca 54720 cctcccactg cagaatagtg gcgtgggctg ccttccccag cacctgctgt atacctagtc 54780 ccattgtaag aggcgggcct gggtgacagg atgcaaagag aagacgccca ggccttgccc 54840 ttcaggcata atcccgggtg cttgaactca gtgccagctg gcacggcaag gtaccaggac 54900 aaatgcgttt ccagccactg tgctgtcgcc acctcccttg attgctgcag ttgattcatt 54960 tattctttat tagtctctca tgttcctaat agccttccca gggaggaatt tgtaaccttg 55020 ttttacagat gcagaacctg agcccctgag aggtgaagtg acttgctcaa ggtcacacgg 55080 agtgagtgtc agggtggcaa ctcacagccg ggtcttccga acccacgtcc tttgtgctac 55140 cgcaggctct ctctgggttc tctgtggact ctatacctag tgccagccct ctgcagggaa 55200 agctctcttc tcagaatcag aggcagctcc actcccaggg ccaggcagtg atcgtgcagc 55260 tctgtgggtc ctctcacttc gcactcactt ccatgctacc ctgttgtgtg ccctggaggt 55320 tcccctgccc catcagcatt ccctcccctg tgccccctgc atgctcttgc acgcgggcac 55380 acgcacacac acacgcacac atgcacgcac acacacccac atgcacatgc ctgcgggttg 55440 cccacatgca tgtgttgccc acatgcaaac acatggacac gcacacacat gcacacatgc 55500 aaacacacgt gcatgcacac acatacacat gcgcacacat acacatgcat gcatgcacat 55560 ggacacccac acacatgcac acacatgtac acacatgcat gtacatacac acacctgtgc 55620 aggggctgag gtgaggcagc tgcacctgaa ggggggaccc acatttccca agggaggaaa 55680 agcagattcc ccagattctg gagcctcaga accctgcctg gagcggggct gaggcaagag 55740 tcaggagacc cctcggtagc tccccacttg ggccttccaa gctacagagc cctgcgccct 55800 ctgggttgat ctctgtcgcc agccccgcat cttcgggagt ttgctttggc ctccagcaac 55860 agtgtagtct ctcccctgcc acctctaggc ttgctgtggg gctgggtacc tggtccagga 55920 aaagacccct agaggtggga cccgggcctc ctccatttat cagccctctt gggcacctcc 55980 tgcatcccct tgacacacag cgctgcccag agcagaggcg gcttctgccc tggtggagct 56040 tccaggtcag cgcaggagag agtaaaagaa agattgcgaa tggaatggaa gcagcctcca 56100 ccaggaagca ggtggagcag caggcgggct gtgagggagg gtctcgatgg catcagggca 56160 ggcttctctc agcttcatca agctgagatg ggaagatgag aagatcagag gaaggttcca 56220 gactgaacca accacaaagg cattccccca gggcatggcc aatgaggaca ggcaggatgt 56280 ggggagacgt gtcagggcgc gctggagagc atgtgactca ggaggcggca gacctgggtc 56340 caggtccagt ccgcatccat atgacttggt tcccgtcacc taacctctga gctacactcc 56400 ctccgtcact caaaaatatg cacagagagt aggcgcagtg gctcctgcct gtagtcccag 56460 ctactcggga ggatcacttg agctcagaag gttgaggctg ccgtgaactg tgattgcacc 56520 accacacacc agcctgggtg acagagtgag accctgtctc tataaataaa taaataaata 56580 aataaggctg ggcacggtgg ctcgtgcctg taatcccagc actttgggag gccgagacag 56640 gcggatcaca tgaggtcggg agttcaagac cagcctggcc aacatggaga aaccccgtct 56700 ctattaaaaa tacaaaatta gctgggtgtg gtggcgggca cctgtgatcc cagctactcg 56760 ggaggttgag gcaggagaat cgtttgaacc cgggaggcag aggttgcggt gagccgagat 56820 cgcgttattg cactccagcc tgggcaacaa gagtgaaact ccgtctctaa ataaataagc 56880 acagggatgt cagggagggt ctgcctcaga ggcgtttggg acaatgacat gagctaatgc 56940 tggtagcacc cctcccatag cccctggcat gaacctctcc cttcctccct gccccctcct 57000 cccctgcttc ttccaccttg tgtggaggcc ctgatactct gtcactgccc attcctggcc 57060 tggctgtagg gtccccgccc ctccttcccc agccttattt aattccaaca gactttatca 57120 ggcacctgct ttggatcagg cctgatactg accaaaatca ggtgtcccaa gttggggaga 57180 ctggccgaga tcctgctggg atggcgcctg gtcagtgatg ctatcccccg accttccttt 57240 ccctttctct gttgcactct ccagcccctg aaatactaga catttgttcc tttactgccc 57300 tcacccccac cctcgctgtc ctgcatagga cagaagttcc tcgggtccgt ctgggtctgt 57360 ctgccaggac acagctgcag gggacaaggg ctatgagggt ggatgtaaag tactgggctg 57420 tggcaggacc ctcctctctc ccactgctgt tcccactgca gccacgacca cctgctgtgc 57480 ctggatggag gaggagtgaa aggcctcatc atcatccagc tcctcatcgc catcgagaag 57540 gcctcgggtg tggccaccaa ggacctgttt gactgggtgg cgggcaccag cactggaggc 57600 atcctggccc tggccattct gcacagtgag ggcggcccct ggggatgggg ccaggcgggg 57660 ctgagacctg tgtcctcaag gggccgagtt tgtcttcagg gctttgcaaa aagagagtgg 57720 aggagtagag gagggctata agcactttga ggaggcaggg gcggggctgc aggtgctgag 57780 gtcccaggct ggaaggagaa gggctgggcc catgggcttg ggtttgggga tcccccgccc 57840 tctggccaca gagggtagag ctggctgtgt gaaattttgg cataggaagc gtttcctatc 57900 gactgcctta acccctcctc ccctcacccc tgcaaagcag gcaccagggg ccacacagcc 57960 tgctgcacat aaggaatccc attcccaggg tgcacgcagc aggcagtctc aggaatgagg 58020 agccactgag cctcaggatg ccagagccct cgcctgcaca cttcatttta ctgggaaatt 58080 ttactgggaa attaagttcc caggtgggga gggatgacgt gaagtcacat agctcatcca 58140 tggggtcgct gggctggagc acagtcccct agactcccag cctggcacct cctaggccac 58200 agggcactgt gtagtctcct ttcactgccg ctttgcgaag gcgcctggct tgctggacag 58260 atagccgggt ggtcagtgtt gtgacggctc ctctgtggag ctgtctgagc ttctaggtgc 58320 tgacaggtgg ggaggggcac tgccagatca gggtcctcag ctccccccaa ccccctggac 58380 attgtcctgg gactcaacat gtctgactgg tgcccctgcc cctgtgctgg tcagattcca 58440 ttttctccag agcgccccca catctggatc ctggtcatgc aaagtcttgg aggtgcccgg 58500 ggaggactcc ccaccctagg gtgggtatca gggcctgtgg gccaggcctg tgttggatgc 58560 tggggatgca gagctgagcc aggcataccc cagcctcagg agcagggggc tcagccagtc 58620 caggaggtgc tggggacaga gtcccatggg cggaggagga gagtcatctg tcaagttgcc 58680 gggacgttag tgtccacggc ggtttattga ctggtatttc atttcctttt gatgattctc 58740 accatagccc tgcggggtga gcagatgggg aaactgagaa ccagagagga tcagccagtg 58800 aattgggcag tcaggactgg gtctccagca gtgtgcccag ctctctaccc cagcctcagg 58860 gtcccagcat cccctctctg gtcttccaca gggtccaagg gcgggcacga ggtctgagca 58920 ggcagactgg gcagagtcct agcttcttct ccaaaagggc cacgtccttc ttcagggacc 58980 atttcttccc catccggggt cttgagactc ccaggggcca tggaggggat ttgagaggtc 59040 tcctggctga tcctccggtg tgggcagatg gcctcctttc agctaccctc tgcttgtaca 59100 tccctggccc tggggaactc actgcctcac gggggaagtg gggcagcttc tccttcctgg 59160 aggtgcagcc ggccccttgg ccctgtctgt tctgaaggct caaaggacag ggctattcct 59220 tgttttctgt gacaaacaac gtgggtctag cttttataag cttttactct tgaggggctc 59280 ttctttaagg aaaataatac aaaatcatga atacaagatg aagtgcagag ccgtggggct 59340 tcagtggttt ttgagtctct ccgttgccca ggctggagtg cagtggtgca tcttggctca 59400 ctgcaacctc cgctcccggg ttcaagcgat tctcctgcct ctcagcctcc cgagtagctg 59460 ggactacagg cgcccgccac cacacctggc gaatttttgt atttttaata gacggggttt 59520 caccatgttg ggcaggctgg tctcaaactc ctgacctcaa gtgatccacc tgcctcagcc 59580 tcccaaagtg ctgggattac aggcgcgagc caccatgtcc agccggaagg aggtttctct 59640 ctgtctgtct gggaggttgc aaaggaaggg cccagaattt gggtttgctt aggcctcggt 59700 aaacccgctt cctcctgcca cctcctgcgt tcaggcgctc tgcaggctgt tctacgggag 59760 ccctgcgtgt ggccgctgca tctccccgcc tctgacccct ttgttcctag gtaagtccat 59820 ggcctacatg cgcggcatgt actttcgcat gaaggatgag gtgttccggg gctccaggcc 59880 ctacgagtcg gggcccctgg aggagttcct gaagcgggag tttggggagc acaccaagat 59940 gacggacgtc aggaaaccca agtaagccct ggcgcactgg ggccgtggcc gcagctgtgc 60000 ccactgtggc tgttccctgg ggacagaggg ctccctgtcc tgctgaggga gggggaagag 60060 cgccccacgc ttgaggggag cagagggaac ccccttccca gggaggcaga gggctggggt 60120 gtggctctca cttgggcatg cagacacctg ctgagcgcag gaggggcgtg gcctggtggg 60180 cctggctctg ccctgcctgg ctctgccctg cccggctctg ccctgccggc tctgccctgc 60240 ctggctctgc cctgcccggc caggggtctg gggcttgcct ggagagagcc tgggccaccc 60300 ttctctgaga ccacctgggc agaggctggg tttttgctgc ccaggcaaaa ggaacccact 60360 ggcctttcaa atgacacggc taaacatttt ctcacatttc agacagcact cgctcccagg 60420 atcggactac taggttataa tcagagggct tcagtcttac ctttaattta aggcctacaa 60480 gacttgaact aatttaagtc agactgaagg agaaagtcac atttccagga aacatttgtt 60540 tgggaagccc aaatcctagg actaagcaat gcatcccgtt cggggctgga tgaatggtgt 60600 gttccaagag gagtgttttg agcatttctg agtgattttt tcatattttg aaagtgagtg 60660 tcccccagtc ttaggggccc ggcatcccct ctgcggtctt ccctagggtc caaggccagg 60720 gaccttcaat cttttttccc actctgtggc ttgtctttct gctctgtggt gtttctaggt 60780 ttttgttttt tgttttttgt tttggttttg gttttggtgg cccatgttct taatgaggta 60840 gataatattt ataaatattt ttcttcttgt agtttgtgat tttttccttt ctttctttct 60900 tttttttgaa acagggtctt gctgtggggc ccatgctgga gtatattgct gtgaacacgg 60960 cttactgcag cctcgacctc ctgggttcaa gcagtcctcc catctcagcc gcctgagtgc 61020 ctgggactac tagcatgtgc caacacatgt attttttgta gagatggggt ttcaccatgt 61080 tgtccaggct agtcttgaac tcctgagctg aggcgatcct cccgccttgg tttcccatta 61140 caggcatcag ccaccatgcc cagcctagtt tgtgcttttt atttctcgtt cataaacccc 61200 tttgctgccc ccaagcagca cacgattctc ctgtgtcgag ctggaaagct ctctagatct 61260 gccttttaca ttgaggtctt ttatcaacct ggaattgatt tttgtatgag ggtgaaatcc 61320 aagatccaat ttaatgtttt ccacgtgatt ggccaatttt tcccagcccc atctgtgaag 61380 tctgtccttc cctccgcttc ctcagcagtg ggtcaagcat ccctctgctc ttggatctgc 61440 tccaggctgt gttttgttcc actggttgga aagctgtctc tgtgcccgga ccacagagca 61500 taaaaaacta ccgctgtgtg gtgagtcctg gtgtctgggt ggcagatcct ccaccttggc 61560 cttcttcaaa aacatcatgg ctattcttgg gggcagtaga gagaagtgga tacaagcata 61620 gaccctgaag tttctacctc ttctcaaaaa taggaatatg ccaggcatgg tggctcaagc 61680 ctgtaatccc agctctttgg gaggctgagg caggaggatc acttgaggcc aggaggtcga 61740 gaccagcctg ggcaacatag tgagaccctc atctctacaa aaaatttaaa actaacaatt 61800 agctgggcat ggtggcacta tggctgtagt cccagctaat tgagaggctg aggtgggagg 61860 atcacttgag ccccggaggc tgaggctaca gtgggctgtg atcccatcac tgcactccag 61920 cctgggcaat agagtgagac cctgtcttaa gacaaaaaaa aaaaaacaaa aacaaacaaa 61980 ctcacaaact ataaaaagaa aaaatattta taaattctac tccattaaga acatgggtcc 62040 atccaaataa ataaataaaa caaaaacacc ataaagggag taaaaagaca agccacagag 62100 tgggagaaaa gatttcgaat ccatacatcc atcaaaggac tcattttcaa aatatgaaaa 62160 gatcattcag aaatgctcaa aatagtaacc accattttac tttctcttcc tatgagttta 62220 actgctccag gtactgttaa gtaaaatttt taggggataa ttgatttgga ccaggattct 62280 gtaccaggcc caacagaaga aacgaatatg gagtcattca tgccaagtga acctagttag 62340 cttaggcgta tacccatgta acaaatagct gagttctggt tagctacaac agctaagctt 62400 taatcaatca tagatggcca cctgattcaa acaaggcaaa ccaattaagc tccacacctc 62460 actttggttt tcagcccatc aacactgcct gaccacgttg caggccagag ttctttgaac 62520 ctattctggt tttgtggcct gcctgactct cagttcatca ataaaagcca attaacatct 62580 ttaaatttgt tgcaatttta tctttgaccg taccttatgt aagtggaatc atacagtatt 62640 tgtctttttg tgactggctt atttctttct tttttttttt ttttgagatg gagtcttgct 62700 ctatcaccca ggctggagtg cagtggcgcg atctcggctc actgcaattt ctgcttccca 62760 ggttcaagtg attctcctgc ctcagcctcc ggagtagctg ggattatagg cgtgcaccac 62820 catgcccggc taattttttg tattttagta gagacagggt ttcaccatgt tggccaggcc 62880 agtctcaaac tcctaacctc gtgatccacc cacctcggcc tcccaaagtg ctgggattac 62940 aggggtgagc caccgtgccc ggccatgact ggcttatttc acttggcatc catgttgtag 63000 cgtgtgtcag aatttccttc ctttttaagg ctgaaaaact ccattgtctg ttggatacac 63060 gttttgcttc tccattcatc cattgatgga tattttagtt gtaaattgac tgctgtcaat 63120 gtcggatgtg caaatacctg tttgaggcct actctctctt actggtgcct tctctagaga 63180 gaattctgat gggttcagtt tgatcatatt ttgtttaaga tttttggcca ggcgcagtgg 63240 ctcacgcttg taatcccagc actttgggag gccgaggtgg gcggatcatg aggtcaggag 63300 atcaagacca cagtgaaacc ccatctctac taaaaataca aaaaattagc cgggcgtggt 63360 ggcgggcgcc tgtagtccca gctactcaga gaggctgagg caggagaatg gcgtgaaccc 63420 gggaggcaga tcttgcagtg agccgagatt gcgccactgt actacagcct gggcaacaga 63480 gtgagactcc gtctcaaaaa aaagattttt gtgtccacgc tcgtgggcga acttgacccg 63540 ttttttccta ttttgggttt tgaggaaata tttcctcttt ttatttttct tggaagagtt 63600 agtataatag tttattttga atttatggta gaacttgata gtaaattctt ctaggcctgg 63660 agtttgcttg gtgggaggat ttttgatgat ggaattaatt tctatagtgg ttgtgagact 63720 gaccactcgg ggtttctaaa tacctttttg agttagtttt ggttaagtga ggtttttcta 63780 ggaatgtgtc catttcatct aaatctttaa gcttattggc ttagagttgt tcaaaatatt 63840 ctgttttttt tttttttttt ttttttttga gacggagttt cactcttatc gcccaggctg 63900 gagtgcaatg acgtgatctc ggctcactgc aacctctgcc tcctgggttc aagcaattct 63960 cttgcttcag cctcccaagt agctgggatt acaggcatgc gcaaccacac ctaatttttg 64020 tattattagt agagacgggg tttccccatg ttggccaggc tggtctcgaa ctcctgacct 64080 caggtgatcc acctgcctcg gcctcccaaa gtgctgggat tacagacatg agccaccatg 64140 cccagccgat tctccttttt ctaacgtctg cagtgtgtgt tggtgcggcc cccttttctt 64200 tcctgatttt gataacagat aacagccatg ttttatcagt cttgcaacag gtttttccat 64260 tggattagcc tttcaaaaaa accaactttt gctgaattgt gcccccaagg ttggaccaat 64320 ctgacactcc tccagcactg ccggagagag cctgttttgc ggggatggtg cggggagaag 64380 cccacctatc ccgaacagag gttggagtgg gcactctgct acccaaggcc ctggggtgtg 64440 aattgtgggg aaagggaaag gtggatgggc gggtggctca gccgtcctcc tgcctcattt 64500 ctctccaggg tgatgctgac agggacactg tctgaccggc agccggctga actccacctc 64560 ttccggaact acgatgctcc agaaactgtc cgggagcctc gtttcaacca gaacgttaac 64620 ctcaggcctc cagctcagcc ctcaggttta aaccatgttt gatgcatcca tgggaagcga 64680 ctggcccgag aggctgtggg gtggtgggtg ggagtcgtgc acttgccatc aggcaccctc 64740 accgtgccac cagcccaccc gctgcactgg tctttattgg ctgaggacag ggattgcggg 64800 ggagctgtca ggcccctggc aggttagaaa agtccctgga aagtcctcag ctgtacctgc 64860 cttccaccag gacgaactag ccagagagtg tggcttcgtg ggtcctgctt ctgaagagtt 64920 cccagcctcc cctcttcccg cacccccagc ccccaacacg cacaccctga gatctggagt 64980 gcatgggttt tatgccagtc ccttgtgcca ctgggccgcc ctccttcccc gcccctagac 65040 cagctggtgt ggcgggcggc ccgaagcagc ggggcagctc ctacttactt ccgacccaat 65100 gggcgcttcc tggacggtgg gctgctggcc aacaacccca cgctggatgc catgaccgag 65160 atccatgagt acaatcagga cctgatccgc aaggtgagtg ccgtaggcca gagggcctgg 65220 acccactgct ccctggagcc aatcctgtgt cagcaaacca tgctagggac cgacccccag 65280 gacagcaggt ggcttttaca cacgcactca ttcatatgtg cacacagggc aaattgatct 65340 cactggaacc tgttccatgg gtgggatctg cctgaagcac tgtgctaagg gggatctgag 65400 gaccactgtg ctctaagtgt gcatgagtgg ggagggcggc atgcatacca tgaacgtgcc 65460 actcgagcca gcctggggac aggacacatg cttgcccttg agctcacctc tgtggataga 65520 cagtcccaca ggaatgcctg tccccacggg ttgccggtgc tgtggtggag gaccgtgagg 65580 cctctgctaa aaggcctggg ggctcggaaa gggctccatg gaacagggga cccatcctga 65640 ggcccttggg gacagagcag tgcagaatca gtgatctgga ggaagagcag cagccctctc 65700 tctgcagagg tacagcagag gctggcagga cccagcatgc taggggaggc cctgtggcgt 65760 ggcgggcggt gggtgaccgg ggtgggcgag cgcggtggct gagctcagac cctggagcca 65820 gggcgcctct gggggtcccc gttctgcctg cactcactgc agctacctaa tggggccatc 65880 atagcgcctg cctggggagg ctgcgaggcc ttggcatgct gttgtctgtg aaatgttcac 65940 agcactgccg ggagcctgag ctgcatcctt gctgctgtgg tttcccgaag ccccagggcg 66000 tcctgggcag cgcagcagag gctctgggca gacagagaag cgccctctag gggccagatc 66060 gggcagggcg ggcttcctgc aggcggcaga gaccgtcaga ctgtcagaca ctagccagtc 66120 taactctcct attgcgtgcg gatggggagc ctgggaccca gagtgggagg catcacccaa 66180 gatcatccag caaatcggca gcagagcttg ttcggaagca aataagagag gcagcggccg 66240 ggcgcagcgg ctcacgcctg taatcccagc actttgggag gccgaggcaa gtggatcatg 66300 aggtcaggag atcgagacca tcctggctaa cacggtgaaa ccctgtctct actaaaaata 66360 caaaaaaatt agccaggcgg gcatagtggc aggagcctgt agtcccagct actcgggatg 66420 ctgaggcagg agaatggtgt gaacccagga ggcggagctt gcagtgagcc gagatcgcgc 66480 cactgcactc cagactggga gacagagcga gactccgtct caaaaaaaaa aaaaaaaagg 66540 cagcacccag ggctcaggtt caagccttag cccctacttc cctaccacgt ggcccctgca 66600 gctgcttccc ggcctgagct tcgctgagag ccctcatttg caaactgggt gatcagtgcc 66660 ctctccacag actggctgga gattaaatga agtaatgatg caaatacctg gcaaatcacc 66720 aacaccaagg aacaccgtct gcctttcctc ccctggccac gacttgactt gggccagcat 66780 cagatggggt gggcgggatc cttcttgcag ctggtgtatg tgtggtcctg ttggtacaca 66840 gatgctgtcc tgagcagatg gcatgtgatg cccgcaccag ccgttctggg gtcacgtgca 66900 agaggctggc tggaaatcat gccaggcatg ctcttgccac cagagacctc ttgccctgtt 66960 ctcatgtgcc ccagagccca gtcttgggcc tccccttgac gctccccttc gtgcccccta 67020 cctgtggaag gtctcttctg actgcccctg tcctgttccc aacagggtca ggccaacaag 67080 gtgaagaaac tctccatcgt tgtctccctg gggacaggga ggtccccaca agtgcctgtg 67140 acctgtgtgg atgtcttccg tcccagcaac ccctgggagc tggccaagac tgtttttggg 67200 gccaaggaac tgggcaagat ggtggtggac tgtgtgagtg tgggcccctc ccccaggcca 67260 cttccctcag ggtctgtagc ccaggtcgat ggcttcccct catcctgtgg ggcctttggt 67320 gttggaggag acagcagggc tctgtccatc gcctttagcc agctggaggg agagagagtc 67380 cacagttcag agtctcagtg cggggcaggg accctcagtg accaccagca ggggggtttt 67440 caaacgcttt ttagctgcgg atccctttct gcaaacatag cgcagtgtag aagcccagaa 67500 tggacaaagg ctccagagcg ggctgggcgc tcaccctgta atcccagcac tttgggaggc 67560 caaggcaggc ggaccacttg agcacagcag tttgagacca gcctgagcaa tataatgaga 67620 ctttgtctct accaaaaaat taaaaactag ctggacatgg tggtgtgtgc ctgtggtccc 67680 agctactcag gaggctgagg tgggaggatt gcttaagccc aagaggcaga ggctgcagtg 67740 agctgtgatc atgccactgc actccagcct gagcaacaga aggagacctt gtctcaaaaa 67800 gaaagaaaga aagagagaga gagagagaga ggagggaggg agggaaactt cagggagagg 67860 ggacatggag gcctgtcgac tcagccgcta ccctttcccc cactcatcat ccctgaaagg 67920 gcactgctga gacctctgcc cctctgattg cacagctggg acagtccctg cagtcccagg 67980 aaggggtttt gcccgagccg ctctgctgct gtgtggctca gcctgactcg aaagagcctg 68040 gggctcccca ggctggggct ccgagagtgc agggcagggc cgggcggggt gcgggccggg 68100 cggggtgtgg gcccggcact caccaaggct gcttctcacc agtgcacgga tccagacggg 68160 cgggctgtgg accgggcacg ggcctggtgc gagatggtcg gcatccagta cttcaggtga 68220 gggctcagcc gccccagccc ttggccccgt gccctggcgt ggtcggactc accgaccttc 68280 ccctcccagc cctagtgtgg actttccctt ccccaagggt ctgctctgtt ccccaagccc 68340 accctggtcc tagctggcgt cccctgccca gcctgagcat cctagggtga ccccctcctc 68400 cctggcccga acagattgaa cccccagctg gggacggaca tcatgctgga tgaggtcagt 68460 gacacagtgc tggtcaacgc cctctgggag accgaggtct acatctatga gcaccgcgag 68520 gagttccaga agctcatcca gctgctgctc tcaccctgag ggtccccagc ctctcaccgg 68580 ccccagctga cctcgtccat tcagcccctg ccaggccaag cccagccact gccctcccgg 68640 gcagatctgg gcccaggcac ctctgagtcc atagaccagg cctgggagaa tgccaagctg 68700 cctgcccgag gctggtcctg aaggcctgtc tcccactaac cccgccttcc agcactttct 68760 gtcattccag gctgggaaag tctagagccc cctttggccc ctttccctga ctgtcaagga 68820 caactgactc ccccatcagc tcaaacatta agggtacccg ggcacaaccg tacccctgcc 68880 cccagcccca gcctccctga gggcctgccg ggctgcctct gccccagccc ccagcaaggg 68940 cactcccagg cttcctggtg ggtgcagccc actccctctg ccctctgctc cgttccctgg 69000 gggctgggac taaagaaatg ggtgtccccc accccatcag ctgggaaagc ccaggccgca 69060 ggagtgggat gcccgttgga ctttgcccct cacactggcc cagcccctca cactgcccca 69120 ccccgagaac cctcagctct caaaggtcac tcctgggagt ttcttcttcc caatggaagt 69180 ggcttaagag ccaaaactga aataaatcat ttggattcaa gttcacctgt gttgtgtgtg 69240 cagtggtgtg agatccacct gttcccctga cccccggctc ccctcggcct tgacccttgg 69300 ccttgaccgg acttttcctt tgtacccgga cactctgttt tccaggatcc ctgggcaggg 69360 acacctcctc tttcctctag gcctccccta aaccggccct tagggtatgc agaggcagct 69420 gccaggccca catacccccg gcaggcccaa ggagggccac aatcttcaac ttcatcttgg 69480 aggaccaggg agagccccat ttgtcaccca gagaggccgg ggactccgcg tggacaggca 69540 gcgcgtgtgc agggccgagg tctgcccagg gctgggcaac tccagcgttt gtgctgctcc 69600 tgctccggca agtggaggag ctgggcgggc cagagcggct gtgggagcca agctcagagg 69660 gcgtggcccc actgtgagcc gcaggcctga ggacagtgag aagtaaagcg gcgttcccct 69720 ccaggggctg ctgctgctgg gtctggcaga gagagtcctg ggggagggca tcgggcagcg 69780 cccgcccagt aaggtgggca atgcctggta taggcaggtg ggagggcggg gccccccagg 69840 gttggggtcc ttgaggaggg ggtgacccag gagggggctc atccacatgg gcacttgtat 69900 cagcccacag ctccaagggg tgaataattg aagggcaggt cacacgagcc tgcccggcac 69960 aggatgcctt ccctgccgga tctggttacg ccccagatca 70000 4 23 DNA Artificial Sequence PCR Primer 4 ggcgtcacca acttgttctc taa 23 5 21 DNA Artificial Sequence PCR Primer 5 cggtcactcg aggtgtagtc g 21 6 23 DNA Artificial Sequence PCR Probe 6 cattccgggt gaaggaggtg gct 23 7 19 DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 3240 DNA Homo sapiens unsure 2770 unknown 10 ctgggggtcc gttccccaac ttcctcggcg ctccggactc ccaagtctcc gccggaccct 60 cctttggata ttcctcgtgt ctccgattct gagagagggg gaagacggtg gggcctcccc 120 acctgccccg cagaag atg cag ttc ttt ggc cgc ctg gtc aat acc ttc agt 172 Met Gln Phe Phe Gly Arg Leu Val Asn Thr Phe Ser 1 5 10 ggc gtc acc aac ttg ttc tct aac cca ttc cgg gtg aag gag gtg gct 220 Gly Val Thr Asn Leu Phe Ser Asn Pro Phe Arg Val Lys Glu Val Ala 15 20 25 gtg gcc gac tac acc tcg agt gac cga gtt cgg gag gaa ggg cag ctg 268 Val Ala Asp Tyr Thr Ser Ser Asp Arg Val Arg Glu Glu Gly Gln Leu 30 35 40 att ctg ttc cag aac act ccc aac cgc acc tgg gac tgc gtc ctg gtc 316 Ile Leu Phe Gln Asn Thr Pro Asn Arg Thr Trp Asp Cys Val Leu Val 45 50 55 60 aac ccc agg aac tca cag agt gga ttc cga ctc ttc cag ctg gag ttg 364 Asn Pro Arg Asn Ser Gln Ser Gly Phe Arg Leu Phe Gln Leu Glu Leu 65 70 75 gag gct gac gcc cta gtg aat ttc cat cag tat tct tcc cag ctg cta 412 Glu Ala Asp Ala Leu Val Asn Phe His Gln Tyr Ser Ser Gln Leu Leu 80 85 90 ccc ttc tat gag agc tcc cct cag gtc ctg cac act gag gtc ctg cag 460 Pro Phe Tyr Glu Ser Ser Pro Gln Val Leu His Thr Glu Val Leu Gln 95 100 105 cac ctg acc gac ctc atc cgt aac cac ccc agc tgg tca gtg gcc cac 508 His Leu Thr Asp Leu Ile Arg Asn His Pro Ser Trp Ser Val Ala His 110 115 120 ctg gct gtg gag cta ggg atc cgc gag tgc ttc cat cac agc cgt atc 556 Leu Ala Val Glu Leu Gly Ile Arg Glu Cys Phe His His Ser Arg Ile 125 130 135 140 atc agc tgt gcc aat tgc gcg gag aac gag gag ggc tgc aca ccc ctg 604 Ile Ser Cys Ala Asn Cys Ala Glu Asn Glu Glu Gly Cys Thr Pro Leu 145 150 155 cac ctg gcc tgc cgc aag ggt gat ggg gag atc ctg gtg gag ctg gtg 652 His Leu Ala Cys Arg Lys Gly Asp Gly Glu Ile Leu Val Glu Leu Val 160 165 170 cag tac tgc cac act cag atg gat gtc acc gac tac aag gga gag acc 700 Gln Tyr Cys His Thr Gln Met Asp Val Thr Asp Tyr Lys Gly Glu Thr 175 180 185 gtc ttc cat tat gct gtc cag ggt gac aat tct cag gtg ctg cag ctc 748 Val Phe His Tyr Ala Val Gln Gly Asp Asn Ser Gln Val Leu Gln Leu 190 195 200 ctt gga agg aac gca gtg gct ggc ctg aac cag gtg aat aac caa ggg 796 Leu Gly Arg Asn Ala Val Ala Gly Leu Asn Gln Val Asn Asn Gln Gly 205 210 215 220 ctg acc ccg ctg cac ctg gcc tgc cag ctg ggg aag cag gag atg gtc 844 Leu Thr Pro Leu His Leu Ala Cys Gln Leu Gly Lys Gln Glu Met Val 225 230 235 cgc gtg ctg ctg ctg tgc aat gct cgg tgc aac atc atg ggc ccc aac 892 Arg Val Leu Leu Leu Cys Asn Ala Arg Cys Asn Ile Met Gly Pro Asn 240 245 250 ggc tac ccc atc cac tcg gcc atg aag ttc tct cag aag ggg tgt gcg 940 Gly Tyr Pro Ile His Ser Ala Met Lys Phe Ser Gln Lys Gly Cys Ala 255 260 265 gag atg atc atc agc atg gac agc agc cag atc cac agc aaa gac ccc 988 Glu Met Ile Ile Ser Met Asp Ser Ser Gln Ile His Ser Lys Asp Pro 270 275 280 cgt tac gga gcc agc ccc ctc cac tgg gcc aag aac gca gag atg gcc 1036 Arg Tyr Gly Ala Ser Pro Leu His Trp Ala Lys Asn Ala Glu Met Ala 285 290 295 300 cgc atg ctg ctg aaa cgg ggc tgc aac gtg aac agc acc agc tcc gcg 1084 Arg Met Leu Leu Lys Arg Gly Cys Asn Val Asn Ser Thr Ser Ser Ala 305 310 315 ggg aac acg gcc ctg cac gtg gcg gtg atg cgc aac cgc ttc gac tgt 1132 Gly Asn Thr Ala Leu His Val Ala Val Met Arg Asn Arg Phe Asp Cys 320 325 330 gcc ata gtg ctg ctg acc cac ggg gcc aac gcg gat gcc cgc gga gag 1180 Ala Ile Val Leu Leu Thr His Gly Ala Asn Ala Asp Ala Arg Gly Glu 335 340 345 cac ggc aac acc ccg ctg cac ctg gcc atg tcg aaa gac aac gtg gag 1228 His Gly Asn Thr Pro Leu His Leu Ala Met Ser Lys Asp Asn Val Glu 350 355 360 atg atc aag gcc ctc atc gtg ttc gga gca gaa gtg gac acc ccg aat 1276 Met Ile Lys Ala Leu Ile Val Phe Gly Ala Glu Val Asp Thr Pro Asn 365 370 375 380 gac ttt ggg gag act cct aca ttc cta gcc tcc aaa atc ggc aga ctt 1324 Asp Phe Gly Glu Thr Pro Thr Phe Leu Ala Ser Lys Ile Gly Arg Leu 385 390 395 gtc acc agg aag gcg atc ttg act ctg ctg aga acc gtg ggg gcc gaa 1372 Val Thr Arg Lys Ala Ile Leu Thr Leu Leu Arg Thr Val Gly Ala Glu 400 405 410 tac tgc ttc cca ccc atc cac ggg gtc ccc gcg gag cag ggc tct gca 1420 Tyr Cys Phe Pro Pro Ile His Gly Val Pro Ala Glu Gln Gly Ser Ala 415 420 425 gcg cca cat cat ccc ttc tcc ctg gaa aga gct cag ccc cca ccg atc 1468 Ala Pro His His Pro Phe Ser Leu Glu Arg Ala Gln Pro Pro Pro Ile 430 435 440 agc cta aac aac cta gaa cta cag gat ctc atg cac atc tca cgg gcc 1516 Ser Leu Asn Asn Leu Glu Leu Gln Asp Leu Met His Ile Ser Arg Ala 445 450 455 460 cgg aag cca gcg ttc atc ctg ggc tcc atg agg gac gag aag cgg acc 1564 Arg Lys Pro Ala Phe Ile Leu Gly Ser Met Arg Asp Glu Lys Arg Thr 465 470 475 cac gac cac ctg ctg tgc ctg gat gga gga gga gtg aaa ggc ctc atc 1612 His Asp His Leu Leu Cys Leu Asp Gly Gly Gly Val Lys Gly Leu Ile 480 485 490 atc atc cag ctc ctc atc gcc atc gag aag gcc tcg ggt gtg gcc acc 1660 Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys Ala Ser Gly Val Ala Thr 495 500 505 aag gac ctg ttt gac tgg gtg gcg ggc acc agc act gga ggc atc ctg 1708 Lys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser Thr Gly Gly Ile Leu 510 515 520 gcc ctg gcc att ctg cac agt aag tcc atg gcc tac atg cgc ggc atg 1756 Ala Leu Ala Ile Leu His Ser Lys Ser Met Ala Tyr Met Arg Gly Met 525 530 535 540 tac ttt cgc atg aag gat gag gtg ttc cgg ggc tcc agg ccc tac gag 1804 Tyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly Ser Arg Pro Tyr Glu 545 550 555 tcg ggg ccc ctg gag gag ttc ctg aag cgg gag ttt ggg gag cac acc 1852 Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu Phe Gly Glu His Thr 560 565 570 aag atg acg gac gtc agg aaa ccc aag gtg atg ctg aca ggg aca ctg 1900 Lys Met Thr Asp Val Arg Lys Pro Lys Val Met Leu Thr Gly Thr Leu 575 580 585 tct gac cgg cag ccg gct gaa ctc cac ctc ttc cgg aac tac gat gct 1948 Ser Asp Arg Gln Pro Ala Glu Leu His Leu Phe Arg Asn Tyr Asp Ala 590 595 600 cca gaa act gtc cgg gag cct cgt ttc aac cag aac gtt aac ctc agg 1996 Pro Glu Thr Val Arg Glu Pro Arg Phe Asn Gln Asn Val Asn Leu Arg 605 610 615 620 cct cca gct cag ccc tca gac cag ctg gtg tgg cgg gcg gcc cga agc 2044 Pro Pro Ala Gln Pro Ser Asp Gln Leu Val Trp Arg Ala Ala Arg Ser 625 630 635 agc ggg gca gct cct act tac ttc cga ccc aat ggg cgc ttc ctg gac 2092 Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro Asn Gly Arg Phe Leu Asp 640 645 650 ggt ggg ctg ctg gcc aac aac ccc acg ctg gat gcc atg acc gag atc 2140 Gly Gly Leu Leu Ala Asn Asn Pro Thr Leu Asp Ala Met Thr Glu Ile 655 660 665 cat gag tac aat cag gac ctg atc cgc aag ggt cag gcc aac aag gtg 2188 His Glu Tyr Asn Gln Asp Leu Ile Arg Lys Gly Gln Ala Asn Lys Val 670 675 680 aag aaa ctc tcc atc gtt gtc tcc ctg ggg aca ggg agg tcc cca caa 2236 Lys Lys Leu Ser Ile Val Val Ser Leu Gly Thr Gly Arg Ser Pro Gln 685 690 695 700 gtg cct gtg acc tgt gtg gat gtc ttc cgt ccc agc aac ccc tgg gag 2284 Val Pro Val Thr Cys Val Asp Val Phe Arg Pro Ser Asn Pro Trp Glu 705 710 715 ctg gcc aag act gtt ttt ggg gcc aag gaa ctg ggc aag atg gtg gtg 2332 Leu Ala Lys Thr Val Phe Gly Ala Lys Glu Leu Gly Lys Met Val Val 720 725 730 gac tgt tgc acg gat cca gac ggg cgg gct gtg gac cgg gca cgg gcc 2380 Asp Cys Cys Thr Asp Pro Asp Gly Arg Ala Val Asp Arg Ala Arg Ala 735 740 745 tgg tgc gag atg gtc ggc atc cag tac ttc aga ttg aac ccc cag ctg 2428 Trp Cys Glu Met Val Gly Ile Gln Tyr Phe Arg Leu Asn Pro Gln Leu 750 755 760 ggg acg gac atc atg ctg gat gag gtc agt gac aca gtg ctg gtc aac 2476 Gly Thr Asp Ile Met Leu Asp Glu Val Ser Asp Thr Val Leu Val Asn 765 770 775 780 gcc ctc tgg gag acc gag gtc tac atc tat gag cac cgc gag gag ttc 2524 Ala Leu Trp Glu Thr Glu Val Tyr Ile Tyr Glu His Arg Glu Glu Phe 785 790 795 cag aag ctc atc cac ctg ctg ctc tca ccc tga gggtccccag cctctcaccg 2577 Gln Lys Leu Ile His Leu Leu Leu Ser Pro 800 805 gccccagctg acctcgtcca ttcagcccct gccaggccaa gcccagccac tgccctcccg 2637 ggcagatctg ggcccaggca cctctgagtc catagaccag gcctgggaga atgccaagct 2697 gcctgcccga ggctggtcct gaaggcctgt ctcccactaa cccccccttc catcactttc 2757 tgtcatgcca ggntgggaaa gtctagagcc ccctttggcc cctttccctg actgtcaagg 2817 acaactgact cccccatcag ctcaaacatt aagggtaccc gggcacaacc gtacccgtgc 2877 ccccagcccc agcctaccct gagggcctgc cgggctgcct ttgccccagc ccccagcaag 2937 ggcattccca ggcttcctgg tgggtgcagc ccaatccctc tgccctctgc tccgttccct 2997 gggggctggg actaaagaaa tgggtgtccc ccaccccatc agctgggaaa gcccaggccg 3057 caggagtggg atgcccgttg gactttgccc ctcacactgg cccagcccct cacactgccc 3117 caccccgaga accctcagct ctcaaaggtc actcctggga gtttcttctt cccaatggaa 3177 gtggcttaag agccaaaact gaaataaatc atttggattc aagttcaaaa aaaaaaaaaa 3237 aaa 3240 11 2296 DNA Homo sapiens 11 cttctagata ttacaacaat cttgtattac tgtgcccact tcctggatga tgggtctgag 60 gttcacagag atcatttgcc caagttccta ctgaaagtgc taaggtattt cctgagactg 120 gctcagggtg tgttgccaga caacaatgca cacattttta acacaatact aaacttagta 180 tattatactt atgttaaaaa tttcggagcc gggcgtggtg gctcacgcct gtaatcccag 240 cacttcggga ggctgaggcg ggcagatcac ttgaggtcag gagttcgaga ccagcctggc 300 caacatggtg aaaccccgtc tctactaaaa atacaaaaaa ttagctgggc gtagtggcgg 360 gcgcctgcaa tctcagctac gcgggaggct gacgcaggag aatcgcttga atccgggagg 420 cggaggttgc agtgagcgag atcgtgccac tgcactccag catggtcgac agagcgagac 480 ttgtctcaaa aaaaaaaaaa aaaaggacat gtggtaattc ccctagggaa tttggcctta 540 gttgtgaaaa ataaaaaagc tataatcaat ttctgttcta tcggaaaaaa atgtattggc 600 tgtttagctg aaattcaaat gtaacttaac atcccgtatt ttttatatgc tacatctggc 660 aaccctggct tgaggctacc tggcaaaaat caaaacaggt tcctccttgt tatgccctat 720 gcctctgttg cctttatttt tatctttacg agcaatcatt aacagtaact tcagactcca 780 ggctctggtc gctttaggag gcggaaggcg gactagggta ggactcctgg ccctcatcaa 840 caggactgcc aagaagccgg gcaccttctg ccccgcacaa acatggccgc gcctcgccgc 900 cgaggattgg ctcccgccga aagctcgtcc tcctggctaa gggagcccct tccattgggc 960 agttaaaaaa aatggcggac cccgcctccc gccgtcctcg ggcgcggggg cggagcctag 1020 aggaggcctg ggacagggcc accagtgatt ggcggagagc ggtggtcagg ccatcacgtg 1080 gcccgaggcc cgtttgtttg cggaagtagg aggaagtaga agtgctgagt aagccgaggt 1140 gagtgacctc gcgggtgggc ggggcctggg ggtccgttcc ccaacttcct cggcgctccg 1200 gactcccaag tctccgccgg accctccttt ggatattcct cgtgtctccg attctgaggc 1260 atgtcctcca ttaaccctta tgtgactccc tgagtgcccc caccttccat cttttcatcc 1320 ccctgcgtcc ccaattccca tcccgagacc gcccgtgtct catctcgaac ttgtggaccc 1380 cagggacccc agcttcgacc ctgagtttct cccctgaacc ccagtctcct ccgtgtgtct 1440 ccctcagtat ccctaacttc ccgagtcaat ccatctcttc tctttccccg accctcagtg 1500 cctctttagg ccccattggc cgtcctaatt ttccttcctg tgctcctcta agtgctcact 1560 tgattcccca cttcacgtcc gtctccacct ttcgtggtgc ctctactcat ttctcactcc 1620 ctgcttctcc tgcctttcct ccctgctttt tttccttgcc tttcctctct cgcgtgcctc 1680 cagacctgcc cccgacatgc tccctccttc ccctcagcct ggccacccca gagtctccca 1740 cccctaatcc tgtgtccacc tcctgctccc acagatgact tggaagctgg tgacagggag 1800 catgtgacca gggtgatcat gggactgggt cggggcaagc ctgtgggtgt gggacaggga 1860 gggcacaggg caaggggacc tgtggccgta tttttaatgg gctacccaca tgaccccaac 1920 gaatacctcc ccctagcttc tgggtctttc cctcacctgc ctcttacccg cccaacagga 1980 tgtcaggcag gttcggtttg gcaacgttga ggctgcaccc ttgggctgaa tgcattgttt 2040 ttgaatgcat tgtttttgtt agagtttctg cctggagttt ggaacctgtt ttctgtccaa 2100 gtctcagttt cctcatctgt gacaggggaa ctgtacctcc tgccctcgta agtctgccat 2160 gaggttcaaa atttgaatga gaaaatgtag aaggtaaagc tttataacca gtatggccct 2220 gtacaaatac atgattttac tgttgggtca taggcagaat tgaaaggagt ggaaagggaa 2280 agggctagaa tgagaa 2296 12 20 DNA Artificial Sequence Antisense Oligonucleotide 12 aaaaccaagc ttgcctgctg 20 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 ggtgtagtcg gccacagcca 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14 cctggctaag agtagatggt 20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 agtatctatg ctatcatagt 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 gctcaatcac aaattgcaaa 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 atacctgtaa tcccaccact 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 tggtctccca aagtgctggg 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 gggaaggcat cctgtgccgg 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 acacgaggaa tatccaaagg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 tcagaatcgg agacacgagg 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 aagaactgca tcttctgcgg 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 tccttcaccc ggaatgggtt 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 atccactctg tgagttcctg 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ctggaagagt cggaatccac 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 actgatggaa attcactagg 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 atgaggtcgg tcaggtgctg 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 cggctgtgat ggaagcactc 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 tcacccttgc ggcaggccag 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 accagctcca ccaggatctc 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 gtctctccct tgtagtcggt 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tggacagcat aatggaagac 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 tgtggatctg gctgctgtcc 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 ctgcgttctt ggcccagtgg 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 cagcatgcgg gccatctctg 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 ctgttcacgt tgcagccccg 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 cggagctggt gctgttcacg 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 tgcagggccg tgttccccgc 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 ttcgacatgg ccaggtgcag 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 agtctgccga ttttggaggc 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 ctggtgacaa gtctgccgat 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 gagctctttc cagggagaag 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 gtgcatgaga tcctgtagtt 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 cgcttctcgt ccctcatgga 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 caggtggtcg tgggtccgct 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 tggatgatga tgaggccttt 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 aggccttctc gatggcgatg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 aatggccagg gccaggatgc 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 tacatgccgc gcatgtaggc 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 aacacctcat ccttcatgcg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 gtgtgctccc caaactcccg 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 ttggtgtgct ccccaaactc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 cagcatcacc ttgggtttcc 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 gtcagcatca ccttgggttt 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 cagtgtccct gtcagcatca 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 cagacagtgt ccctgtcagc 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 gctgccggtc agacagtgtc 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 tggagcatcg tagttccgga 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 accagctggt ctgagggctg 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 taagtaggag ctgccccgct 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 tgggtcggaa gtaagtagga 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gggttgttgg ccagcagccc 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 tactcatgga tctcggtcat 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 tcctgattgt actcatggat 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 cccagggaga caacgatgga 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 ggacggaaga catccacaca 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 aacagtcttg gccagctccc 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 atcttgccca gttccttggc 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 aacagtccac caccatcttg 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 gatccgtgca acagtccacc 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 tggatccgtg caacagtcca 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 gccgaccatc tcgcaccagg 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 agtactggat gccgaccatc 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 tctgaagtac tggatgccga 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 actgacctca tccagcatga 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 ctcggtctcc cagagggcgt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 agatgtagac ctcggtctcc 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 ctcatagatg tagacctcgg 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 cggtgctcat agatgtagac 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 ggaccctcag ggtgagagca 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 caggcctggt ctatggactc 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 accagcctcg ggcaggcagc 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 cacccaccag gaagcctggg 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 catttcttta gtcccagccc 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 cggcctgggc tttcccagct 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 atcccactcc tgcggcctgg 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 caggagtgac ctttgagagc 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 ttgaatccaa atgatttatt 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 tactggttat aaagctttac 20 

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding Phospholipase A2, group VI (Ca2+-independent), wherein said compound specifically hybridizes with and inhibits the expression of Phospholipase A2, group VI (Ca2+-independent).
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 60, 61, 63, 64, 65, 66, 67, 68, 70, 73, 75, 76, 77, 79, 80, 82, 84, 87, 88 or
 89. 4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding Phospholipase A2, group VI (Ca2+-independent).
 12. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 13. The composition of claim 12 further comprising a colloidal dispersion system.
 14. The composition of claim 12 wherein the compound is an antisense oligonucleotide.
 15. A method of inhibiting the expression of Phospholipase A2, group VI (Ca2+-independent) in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Phospholipase A2, group VI (Ca2+-independent) is inhibited.
 16. A method of treating an animal having a disease or condition associated with Phospholipase A2, group VI (Ca2+-independent) comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Phospholipase A2, group VI (Ca2+-independent) is inhibited.
 17. The method of claim 16 wherein the disease or condition is caused by abnormal apoptosis.
 18. The method of claim 16 wherein the disease or condition is diabetes mellitus.
 19. The method of claim 16 wherein the disease or condition is a hyperproliferative condition.
 20. The method of claim 19 wherein the hyperproliferative condition is cancer. 